Method of making an assay compound

ABSTRACT

An assay compound or a salt thereof for assaying the activity of an enzyme inside a metabolically active whole cell is disclosed. The assay compound includes a leaving group and an indicator group. The leaving group is selected from the group comprising amino acids, peptides, saccharides, sulfates, phosphates, esters, phosphate esters, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures thereof. The indicator group is selected from compounds which have a first state when joined to the leaving group, and a second state when the leaving group is cleaved from the indicator group by the enzyme. Preferably, the indicator compounds are rhodamine 110, rhodol, and fluorescein and analogs of these compounds. A method of synthesizing the compound as well as methods of using these compounds to measure enzyme activity are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to cytoenzymology, and moreparticularly reagents for use in cytoenzymology as well as productionand use of these reagents.

2. Description of the Background Art

Cytoenzymology is the study of enzymes as they function on and withincells. Previously, the study of enzymatic activity within cells has beenpursued primarily by two indirect methods. According to a first method,the cell membrane is broken to create a cytosol of cellular componentsincluding the enzyme-which is the object of study. Various tests arethen performed to determine the activity of the enzyme, which tests canbe performed on the cytosol or on the purified enzyme. According to asecond method, the enzyme activity is determined from the study ofextra-cellular events, such as the presence or lack of the products ofenzyme activity.

According to the first method, various tests are performed to determineenzyme activity in the cytosol. One such test is to provide a substratethat is recognized by the enzyme, with a fluorescent compound which willundergo a detectable change when the substrate, or "leaving group", iscleaved from the compound by the enzyme. Mangel et al., U.S. Pat. Nos.4,557,862 and 4,640,893, disclose rhodamine 110-based derivatives asfluorogenic substrates for proteinases. These compounds have the generalformula:

    (Cbz-peptide-NH).sub.2 -rhodamine 110

where the peptide includes known amino acids or amino acid derivatives,and "Cbz" refers to the blocking group benzyloxycarbonyl. When the aminogroups of rhodamine 110 are blocked the compound is "quenched", and isrelatively colorless and non-fluorescent. Cleavage of one of thepeptides from the non-fluorescent bisamide substrate results in a3500-fold increase in fluorescence intensity.

The rhodamine 110 substrates of Mangel et al. have been utilized toconduct cytoenzymological studies. G. Rothe et al., Biol. Chem.Hoppe-Seyler, 373, 544-547 (1992) describe the analysis of proteinaseactivities using the substituted peptide-rhodamine 110 derivatives ofMangel et al. Moreover, G. Valet et al, Ann NY Acad Sci, 667, 233-251(1993), disclose the study of white cell and thrombocyte disorders withthe rhodamine 110 derivatives of Mangel et al. The methods of Rothe andValet have been used to conduct cytoenzymological studies on theactivity of enzymes with cells, but the compounds utilized by Rothe andValet are not suitable for the study of the activity of intracellularenzymes in vital cells. The Mangel et al. compounds cannot beefficiently solubilized and transmitted through the cell membrane in amanner which will produce a reliable assay. In addition, the Cbz groupin the Mangel et al. compound is not recognized by the enzyme's activesites. Further, Mangel, et al., disclose the removal of thecarbobenzyloxy group by treating the blocked peptide-indicator compoundwith 30% hydrobromide acid in acetic acid. However, the bromide salt islethal to the cell and does not permit an assay for a metabolicallyactive cell.

I. Mononen, et al., Clin. Chem., 40 (3), 385-388 (1994), describe theenzymatic diagnosis of aspartylglycosaminuria by the fluorometric assayof glycosylasparaginase in serum, plasma, and lymphocytes. The study wasconducted on cytosols, and not whole cells, and utilized anasparagine-substituted 7-amino-4-methylcoumarin.

Dead or metabolically inactive cells can have as little as approximatelyone-quarter the enzymatic activity of living cells, Watson, J., "EnzymeKinetic Studies in Cell Population Using Fluorogenic Substrates and FlowCytometric Techniques", Cytometry, 1(2), p. 143 (1980). Further, becauseenzymes are frequently bound in highly organized enzyme pathways, thedisruption and death of the cell can greatly affect enzyme activity.Current assays therefore have limited utility for determining enzymeactivity in a living or metabolically active whole cell.

U.S. Pat. No. 5,070,012 to Nolan et al., describes a method ofmonitoring cells and trans-acting transcription elements. This method,however, is not designed for the monitoring of enzymes which areendogenous to the cell being tested. Rather, in this method a hypotonicsolution is used to increase the permeability of the cell membranethereby allowing an exogenous enzyme and other reagents (including afluorogenic substrate) to be introduced into the cell. However, thesesevere hypotonic conditions significantly alter the normal state of thecell. The fluorogenic substrate described in this patent (fluorosceindigalactopyranoside) contains significant amounts of fluorescentimpurities and must be bleached with a laser prior to use.

SUMMARY OF THE INVENTION

The present invention relates to an assay reagent for determining theactivity of an enzyme in a metabolically active whole cell, said assayreagent comprising at least one water soluble assay compound having theability to pass through a cell membrane or a water solublephysiologically acceptable salt thereof having the ability to passthrough a cell membrane, said assay compound having a leaving groupselected for cleavage by an enzyme to be analyzed and a fluorogenicindicator group being selected for its ability to have a non-fluorescentfirst state when joined to the leaving group, and a fluorescent secondstate excitable at a wavelength above 450 nm when the leaving group iscleaved from the indicator group by the enzyme, said assay reagenthaving a fluorescence less than the auto-fluorescence of a metabolicallyactive cell and being stable for a minimum of 30 days when stored atwherein said stability is defined as the compound having an increase inbackground fluorescence of ≦10%.

The present invention also relates to an assay reagent for determiningthe activity of an enzyme in a metabolically active whole cell, saidassay reagent comprising at least one water soluble salt of an assaycompound having the ability to pass through a cell membrane, said assaycompound having a leaving group selected for cleavage by an enzyme to beanalyzed and a fluorogenic indicator group being selected for itsability to have a first non-fluorescent state when joined to the leavinggroup, and a second fluorescent state excitable at a wavelength above450 nm when the leaving group is cleaved from the indicator group by theenzyme, and said assay reagent having a fluorescence less than theauto-fluorescence of a metabolically active cell.

The present invention also relates to an assay reagent composition fordetermining the activity of an enzyme in a metabolically active wholecell, said assay reagent comprising at least one water soluble assaycompound having the ability to pass through a cell membrane or a watersoluble physiologically acceptable salt thereof having the ability topass through a cell membrane, said assay compound having a leaving groupselected for cleavage by an enzyme to be analyzed and a fluorogenicindicator group being selected for its ability to have a non-fluorescentfirst state when joined to the leaving group, and a fluorescent secondstate excitable at a wavelength above 450 nm when the leaving group iscleaved from the indicator group by the enzyme, and at least oneadditive selected from the group consisting of a buffer, an enzymecofactor, an enzyme modulator, an enzyme inhibitor, an enzyme activator,a solubilizing component for said assay reagent, and a retentioncomponent for said assay reagent or products thereof, said assay reagenthaving a fluorescence less than the auto-fluorescence of a metabolicallyactive cell.

This invention also relates to a method to produce an assay reagent fordetermining the activity of an enzyme in a metabolically active wholecell, in which the cell is contacted with the assay reagent. In a broadaspect, the invention relates to a method to make an assay compound forassaying the activity of an enzyme inside a metabolically active wholecell, said assay compound comprising an indicator group and a leavinggroup, said leaving group being selected for cleavage by said enzyme,comprising reacting a compound containing a leaving group selected fromthe group consisting of amino acids, peptides, saccharides, sulfates,phosphates, esters, phosphate esters, nucleotides, polynucleotides,nucleic acids, pyrimidines, purines, nucleosides, lipids and mixturesthereof and a blocking group, with an agent to form an intermediatecomplex containing a leaving group and a blocking group, reacting theintermediate complex with a compound containing an indicator group toform a reaction product; separating the reaction product from sidereaction products, by-products and starting materials, removing blockinggroups from the reaction product to obtain an assay compound having anindicator group and leaving group, optionally reacting the intermediatecompound having an indicator group and leaving group with an acid orbase to form a physiologically acceptable salt of said assay compoundfor assaying the activity of an enzyme inside a metabolically activewhole cell, and purifying the assay compound or the physiologicallyacceptable salt thereof.

More specifically, the present invention is further related to a methodfor making an assay compound for assaying the activity of an enzymeinside a metabolically active whole cell, said assay compound comprisingan indicator group and a leaving group, said leaving group beingselected for cleavage by said enzyme, comprising reacting a compoundcontaining a leaving group selected from the group consisting of aminoacids, peptides, saccharides, esters, nucleotides, lipids and mixturesthereof, and a blocking group with an agent to form an intermediatecomplex containing a leaving group and a blocking group, reacting theintermediate complex with a compound containing an indicator group toform a reaction product, separating the reaction product from sidereaction products, by-products and starting materials, removing theblocking group from the reaction product to obtain an assay compoundhaving an indicator group and leaving group, and purifying the assaycompound.

In another embodiment, the present invention is further related to amethod for making an assay compound in a salt form for assaying theactivity of an enzyme inside a metabolically active whole cell, saidassay compound comprising an indicator group and a leaving group, saidleaving group being selected for cleavage by said enzyme, comprisingreacting a compound containing a leaving group selected from the groupconsisting of amino acids, peptides, phosphates, sulfates, esters,nucleotides and mixtures thereof, and a blocking group with an agent tofor man intermediate complex containing a leaving group and a blockinggroup, reacting the intermediate complex with a compound containing anindicator group to form a reaction product, separating the reactionproduct from side reaction products, by-products and starting materials,removing the blocking group from the reaction product to obtain an assaycompound having an indicator group and leaving group, reacting theintermediate compound having an indicator group and leaving group withan acid or a base to form a physiologically acceptable salt of saidassay compound for assaying the activity of an enzyme inside ametabolically active whole cell, and purifying the physiologicallyacceptable salt of said assay compound.

The assay reagent has at least one assay compound having an indicatorgroup and a leaving group. The leaving group is selected for cleavage bythe enzyme to be assayed. The indicator group is in a first state whenjoined to the leaving group (e.g. the indicator is non-fluorescent), andis in a second state when the leaving group is cleaved from theindicator group by the enzyme (e.g. the indicator group is fluorescent).

The present invention also relates to a method for determining theactivity of an endogenous enzyme in a metabolically active whole cell,comprising contacting a metabolically active whole cell with an assayreagent under conditions which allow said assay reagent to pass intosaid metabolically active whole cell, said assay reagent having at leastone assay compound having the ability to pass through a cell membrane ora physiologically acceptable salt thereof having the ability to passthrough a cell membrane, said assay compound comprising a fluorogenicindicator group and a leaving group, said leaving group being selectedfor cleavage by said enzyme, said indicator group being in anon-fluorescent first state when joined to said leaving group, and beingin a fluorescent second state excitable at a wavelength above 450 nmwhen said leaving group is cleaved from said indicator group by saidenzyme for a period of time sufficient for said assay reagent to betransferred into said cell and for said leaving group to be cleavedinside said cell from said indicator group by said enzyme, exposing saidcell to light having a wavelength above 450 nm, and measuringfluorescence of said cell.

The present invention also relates to a method for detecting anabnormality in the activity of an enzyme in a metabolically active wholecell, comprising (a) contacting a reference, metabolically active wholecell having a normally functioning enzyme with a medium containing anassay reagent, said assay reagent having at least one water solubleassay compound having the ability to pass through a cell membrane or awater soluble physiologically acceptable salt thereof having the abilityto pass through a cell membrane, said assay compound comprising afluorogenic indicator group and a leaving group, said leaving groupbeing selected for cleavage by said enzyme, said indicator group beingin a non-fluorescent first state when bonded to said leaving group, andbeing in a fluorescent second state excitable at a wavelength above 450nm when said leaving group is cleaved from said indicator group by saidenzyme, for a period of time sufficient for said assay compound to betransferred into said cell and for said leaving group to be cleavedinside said cell from said indicator group by said enzyme, (b) sensingfor said fluorescent second state of said indicator group for thereference, metabolically active whole cell to produce reference results,(c) contacting a test, metabolically active whole cell with said mediumfor said period of time, (d) sensing for said fluorescent second stateof said indicator group for the test, metabolically active whole cell toproduce test results, and (e) comparing the reference results ofreference test, metabolically active whole cell in said step (b) withthe test results obtained from said test metabolically active whole cellin said step (d).

The present invention also relates to a method of performing an assayfor detecting the presence of a disease comprising (a) contacting atest, metabolically active whole cell with an assay reagent, said assayreagent containing at least one water soluble assay compound or watersoluble physiologically acceptable salt thereof having a fluorogenicindicator group and a leaving group, said leaving group being selectedfor cleavage by a enzyme the activity of which changes with the presenceof the disease, said indicator group being in a non-fluorescent firststate when bonded to said leaving group, and being in a fluorescentsecond state excitable at a wavelength above 450 nm when said leavinggroup is cleaved from said indicator group by said enzyme for a periodof time at least sufficient for said assay compound to be transferredinto said cell and for said leaving group to be cleaved inside said cellfrom said indicator group by said enzyme, (b) sensing for saidfluorescent second state of the indicator group for the test,metabolically active whole cell to produce test results, and (c)comparing the test results of said test metabolically active whole cellwith reference results obtained from at least one of a diseasedreference cell and a non-diseased reference cell.

The present invention also relates to a method for detecting anabnormality in the activity of an enzyme in a metabolically active wholecell, comprising (a) contacting a plurality of reference, metabolicallyactive whole cells, each having at least one normally functioning enzymewith a medium containing an assay reagent, said assay reagent having atleast one water soluble assay compound having the ability to passthrough a cell membrane or a water soluble physiologically acceptablesalt thereof having the ability to pass through a cell membrane, saidassay compound comprising a fluorogenic indicator group and a leavinggroup, said leaving group being selected for cleavage by one of said atleast one normally functioning enzymes, said indicator group being in anon-fluorescent first state when bonded to said leaving group, and beingin a fluorescent second state excitable at a wavelength above 450 nmwhen said leaving group is cleaved from said indicator group by the oneof said at least one normally functioning enzyme, for a period of timesufficient for said assay compound to be transferred into each of saidplurality of reference, metabolically active whole cells for each of theat least one normally functioning enzymes for each of said plurality ofreference, metabolically active whole cells to produce a matrix ofreference results and for said leaving group to be cleaved inside ofeach of said plurality of reference, metabolically active whole cellsfrom said indicator group by the one of said at least one normallyfunctioning enzymes, (b) sensing for said fluorescent second state, ofsaid indicator group for each of the at least one normally functioningenzymes for each said plurality of reference, metabolically active wholecells to produce a matrix of reference results, (c) contacting aplurality of test, metabolically active whole cells, each having atleast one normally functioning enzyme with said medium for said periodof time, (d) sensing for said fluorescent second state of said indicatorgroup for each of the at least one normal functioning enzyme for each ofsaid plurality of test, metabolically active whole cells to produce amatrix of test results, and (e) comparing the matrix of test results ofsaid plurality of test, metabolically active whole cells in said step(d) with the matrix of reference results obtained from said plurality ofreference, metabolically active whole cells in said step (b).

The present invention also relates to a method of performing at leastone or more assays for detecting the presence of a disease comprising(a) contacting at least one or more test, metabolically active wholecells with one or more assay reagents, said assay reagent containing atleast one water soluble assay compound or water soluble physiologicallyacceptable salt thereof having a fluorogenic indicator group and aleaving group, said leaving group being selected for cleavage by one ofsaid at least one normally functioning enzyme the activity of whichchanges with the presence of the disease, said indicator group being ina non-fluorescent first state when bonded to said leaving group, andbeing in a fluorescent second state excitable at a wavelength above 450nm when said leaving group is cleaved from said indicator group by theone of said at least one normally functioning enzyme for a period oftime at least sufficient for said assay compound to be transferred intosaid cell and for said leaving group to be cleaved inside said cell fromsaid indicator group by said enzyme, (b) sensing for said fluorescentsecond state of the indicator group for the test, metabolically activewhole cell to produce a matrix of test results, and (c) comparing thematrix of test results of said test metabolically active whole cell witha matrix of reference results obtained from at least one of a diseasedreference cell and a non-diseased reference cell.

The cell is contacted with the assay compound for a period of timesufficient for the assay reagent to be transferred into the cell and forthe leaving group to be cleaved from the indicator group by the enzyme.The assay compound is capable of or enabled to pass through the membraneof the cell so that the enzyme, if present and active, can cleave theleaving group thereby forming the indicator compound which can be sensedfrom outside the cell.

The cell is then sensed for the first state or second state or bothfirst and second states of the indicator group.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

There are shown in the drawings embodiments which are presentlypreferred, it being understood, however, that the invention is notlimited to the precise instrumentalities and arrangements shown,wherein:

FIGS. 1A, 1B, 1C and 1D are flow charts of four assay protocolsaccording to the invention;

FIGS. 2A, 2B, 2C and 2D are charts illustrating the use of salts toenhance specificity;

FIGS. 3A, 3B, 3C and 3D are graphs illustrating the use of inhibitors inthe reagent formula;

FIGS. 4A and 4B are photomicrographs of normal Ficoll preparedlymphocytes and acute lympholytic Ficoll prepared lymphocytes,respectively, which illustrate use of an assay according to theinvention to determine immune competence and the difference in enzymeactivity between normal lymphocytes and acute lymphocytic leukemialymphocytes.

FIGS. 5A and 5B are color photomicrographs which illustrate use of anassay according to the invention to provide an indication of leukemia;

FIGS. 6A and 6B are color photomicrographs which illustrate use of anassay to provide an indication of sepsis;

FIG. 7 is a color photomicrograph which illustrates the use of an assayto provide an indication of the metastatic potential of tumors;

FIGS. 8A and 8B are color photomicrographs which illustrate the use ofassays to monitor drug treatment;

FIG. 9 is a color photomicrograph which illustrates the use of assays toprovide an indication of macrophage. activation;

FIGS. 10A and 10B are graphs illustrating the storage stability of amonopeptide-TFA salt derivative of rhodamine 110;

FIGS. 11A, 11B, 11C, 11D, 11E and 11F are graphs illustrating thestorage stability of TFA salts of dipeptide derivatives of rhodamine110;

FIGS. 12A, 12B, 12C and 12D are graphs illustrating the storagestability of acetate and tartrate salts of the Leu-Gly peptidederivative of rhodamine 110;

FIGS. 13A and 13B are graphs illustrating the storage stability offree-amine peptide derivatives of rhodamine 110;

FIG. 14A illustrates reducing a full covariance data matrix to a reducedcovariance data matrix of strongly contributing factors, by eigenvectoranalysis;

FIG. 14B illustrates the prediction of disease probabilities from thereduced covariance data matrix using Non-Negative Least Squares (NNLS)analysis;

FIG. 15 illustrates a full covariance data matrix being fed to a NeuralNetwork to predict disease probabilities;

FIG. 16 illustrates a comparison of disease probabilities byperforming 1) NNLS analysis on the full covariance data matrix, 2) NNLSanalysis on a reduced covariance data matrix defined by eigenvectors 1and 2, including 11 substrates, 3) NNLS analysis on a reduced covariancedata matrix defined by eigenvector 1 above, including 6 substrates, and4) squared deviation from the mean analysis on 6 substrates;

FIGS. 17A-17C are graphs of the ratio of a disease to the mean of thenormal of all patients with the disease; and

FIGS. 18A-18F illustrate a progression of disease during treatment andmonitoring a return to normalcy and

FIG. 18G is a summary of the data illustrated in FIGS. 18A-18F.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Assay Compound

According to the present invention, an assay reagent is manufactured fordetermining the activity of an enzyme in a metabolically active wholecell. The assay reagent must be compatible with the cell such that thecell will remain metabolically active for at least the duration of theassay.

The assay reagent comprises at least one assay compound which is capableof passing through the cell wall. The assay compound must be smallenough that it can be transmitted into the cell. An assay compoundhaving a molecular weight of less than about 5,000 is presentlypreferred.

The assay compound contains a leaving group and an indicator group. Theleaving group is selected for cleavage by the enzyme to be analyzed. Theindicator group is selected for its ability to have a first state whenjoined to the leaving group, and a second state when the leaving groupis cleaved from the indicator group by the enzyme. The indicator groupis preferably excitable (caused to fluoresce) at a wavelength about thevisible range, for example, at wavelength between about 450 to 500nanometers (nm). The indicator group will usually emit in the range ofabout 480 to 620 nm, preferably 500 to 600 nm and more preferably 500 to550 nm. Auto-fluorescence of the cell is most prevalent below about 500nm.

Indicator groups

The indicator group is preferably derived from fluorogenic andchemiluminescent compounds. The indicator group should be quenched whenjoined to the leaving group. The term quenched means that the indicatorgroup has almost no fluorescence or chemiluminescence when joined to theleaving group. When the leaving group is separated from the indicatorgroup, the resulting indicator compound will have a fluorescence.

Suitable fluorogenic indicator compounds include xanthine compounds.Preferably, the indicator compounds are rhodamine 110; rhodol; andfluorescein. These compounds have the following structures: ##STR1##

In addition, derivatives of these compounds which have the 4' or 5'carbon protected are acceptable indicator compounds. Preferred examplesof the derivative compounds include 4'(5')thiofluorescein,4'(5')-aminofluorescein, 4'(5')-carboxyfluorescein,4'(5')-chlorofluorescein, 4'(5')-methylfluorescein,4'(5')-sulfofluorescein, 4'(5')-aminorhodol, 4'(5')-carboxyrhodol,4'(5')-chlororhodol, 4'(5')-methylrhodol, 4'(5')-sulforhodol;4'(5')-aminorhodamine 110, 4'(5')-carboxyrhodamine 110,4'(5')-chlororhodamine 110, 4'(5')-methylrhodamine 110,4'(5')-sulforhodamine 110 and 4'(5')thiorhodamine 110. "4'(5')" meansthat at the 4' or 5' position the hydrogen atom on the carbon atom issubstituted with a specific organic group or groups as previouslylisted.

Leaving groups

The leaving group is selected according to the enzyme that is to beassayed. The leaving group will have utility for assaying many kinds ofcellular enzymes, including proteases, glycosidases, glucosidases,carbohydrases, phosphodiesterases, phosphatases, sulfatases,thioesterases, pyrophosphatases, lipases, esterases, nucleotidases andnucleosidases. For the purposes of this disclosure the termcarbohydrases includes all enzymes which will hydrolyze a carbohydrate.Enzymes which do not recognize and cleave a leaving group, such asdehydrogenases and kinases, are not suitable for assays according to theinvention. The enzymes to be measured can be those which are present invarious cell preparations, enzymes found in cytosols, cell surfaceenzymes, cytoplasmic enzymes and cell nucleus (nuclear) enzymes.However, as will be discussed herein, the assay compounds areparticularly useful for detecting intracellular enzymes in living cells.

The leaving group is selected from amino acids, peptides, saccharides,sulfates, phosphates, esters, phosphate esters, nucleotides,polynucleotides, nucleic acids, pyrimidines, purines, nucleosides,lipids and mixtures thereof. For example, a peptide and a lipid leavinggroup can be separately attached to a single assay compound such asrhodamine 110.

Other leaving groups suitable for the enzyme to be assayed can bedetermined empirically or obtained from the literature. See, forexample, Mentlein, R., Staves, R., Rix-Matzen, H. and Tinneberg, H. R.,"Influence of Pregnancy on Dipeptidyl Peptidase IV Activity (CD26Leukocyte Differentiation Antigen) of Circulating Lymphocytes", Eur. J.Clin. Chem. Clin. Biochem., 29, 477-480 (1991); Schon, E., Jahn, S.,Kiessig, S., Demuth, H., Neubert, K., Barth, A., Von Baehr, R. andAnsorge, S., Eur. J. Immunol., 17, 1821-1826 (1987); Ferrer-Lopez, P.,Renesto, P., Prevost, M., Gounon, P. and Chignard, M., "Heparin InhibitsNeutrophil-Induced Platelet Activation Via Cathepsin", J. Lab Clin. Med.119(3), 231-239 (1992); and Royer, G. and Andrews, J., "ImmobilizedDerivatives of Leucine Aminopeptidase and Aminopeptidase M.", The J. ofBiological Chemistry, 248(5), 1807-1812 (1973). These references arehereby incorporated by reference in their entirety. Various leavinggroups are shown in Table 1.

    TABLE 1                                                                          -     pH.sup.1                                                                   SUB.  RANGE       IONIC FUNCTION                                               CONC.  (exper- pH.sup.2     TIME STRG OR                                     ENZYME SUBSTRATES (mM) BUFFER iment) lit COFACTOR MODULATER INHIBITOR         mOSM (MIN) (μ) INDICATION CD                                                 AminoPept A (H--L--Asp).sub.2 --Rho 110-2TFA 4.8  Hanks 7.5 7.0 1-5 mM  1     0 mM 280-310  5                                                                 .1530                                                          CaCl.sub.      2  CaCl.sub.2                                                                           Amistatin I                                                             (H--L--Glu).sub.2 --Rho 110-2TFA  Hanks  7.0    280-310                       3              AminoPept B (H--L--Arg).sub.2 --Rho 110-4TFA 3.2  Hanks        8.0 ± .2 7.5 NaCl  10.sup.-4 M 280-310                                       3                                    137 mM  Bestatin                           (H--L--Cys).sub.2 --Rho 110-2TFA  Hanks  7.5 1 mM DTT   280-310  3                AminoPept M (H--L--Ala).sub.2 --Rho 110-2TFA 6.4  Hanks 7.0 7.5           10.sup.-4 M 280-310                                                             5 0.1530 Leukemia CD13                                       Bestatin             (H--L--Ala).sub.2 -4'chloro--Rho 110-2TFA 6.4  Hanks 7.0 7.5               10.sup.-4 M 280-310                                                             5 0.1530 Leukemia CD13                                       Bestatin             H--L--Leu Rhodol--TFA 2.4-3.2 Hanks 5.0-7.0 7.5   1.5 mM 1,10-             280-310                                                                         1 0.1494                                                                      (H--L--Leu).sub.2 4'chloro-Rho 110-2TFA 2.4-3.2 Hanks 5.0-7.0 7.5              Phenalthroline 280-310                                                          1 0.1494                                           (H--L--Leu).sub.2          Rho 110-2TFA 2.4-3.2 Hanks 5.0-7.0 7.5    280-310  1 0.1494  CD13                       (H--L--Met).sub.2 Rho 110-2TFA  Hanks  7.5   56 mM 2,2 280-310        5   CD13                                                                                Dipyridyl I                                                             (H--Gly).sub.2 Rho 110-2TFA 6 Hanks 7.0-7.5 7.5  1 mM DTE  280-310  5        0.1526  CD13                                                                     (H--Gly).sub.2 4'chloro-Rho 110-2TFA 6 Hanks 7.0-7.5 7.5  1 mM DTE           280-310                                                                         5 0.1526  CD13                                                                AminoPept N (H--L--Pro).sub.2 --Rho 110-2TFA 6 Hanks  7.5  1 mM DTE            280-310                                                                         5 0.1526  CD13                                                                (H--L--Lys).sub.2 Rho 110-4TFA 2.4  Hanks 7.5 ± .2 7.5    280-310  5        0.1490  CD13                                                                     (H--L--Lys).sub.2 Rho 110-4TFA 2.4  Hanks 5.5 ± .2 7.5    280-310         5 0.1490                                                                         (H Gly).sub.2 Rho 110-2TFA 6.4  Hanks 5.5-6.0 7.5    280-310  5                       (H--L--Ser).sub.2 Rho 110-2TFA 2.4  Hanks 5.0-6.5 7.5                 280-310                                                                         5 0.1506                                                         Neg          Pro (H--L--Pro).sub.2 Rho 110-2TFA 6 Hanks  7.5    280-310  5 0.1526                Control                                                                    DPP I (H--L--Pro--Arg).sub.2 Rho 110-4TFA 6 MES 5.0-6.5 6.5  ImM DTT          280-310 10                                                                       (H--Gly--Arg).sub.2 Rho 110-4TFA  MES 5.0-6.5 6.5  ImM DTT  280-310 10        DPP II (H--L--Lys--Ala).sub.2 Rho 110-4TFA 2.0  MES 6.5 ± .5 6.5           MgCl.sup.2 Zn DTE Bestatin 280-310 10 0.1019                                     (H--L--Lys--Ala).sub.2 Rho 2.0  Mes 6.5 ± .5 6.5 MgCl.sup.2 Zn DTE        Bestatin 280-310 10 0.1019                                                       110-Sulfo-4TFA                                                                 (H--L--Lys--Pro).sub.2 Rho 110-4TFA  MES 5.5 6.5 MgCl.sup.2 Zn DTE           Bestatin 280-310 10                                                              (H--L Ala--Pro).sub.2 Rho 110-2TFA  MES 5.5 6.5    280-310 10                  (H--L Lys--Ala--Lys--Ala).sub.2 Rho 3.2  MES  6.5 MgCl.sup.2 Zn DTE          Bestatin 280-310 10 0.1229                                                       110-6TFA                                                                      DPP IV (H--L Ala--Pro).sub.2 Rho 110-2TFA        280-310 10   CD26                   (H--Gly--Pro).sub.2 Rho-2TFA 2.4  Gly--NaOH 7.5 ± .5 8.7             Gly--Pro 280-310 10 0.1449  CD26                                                        1.8 mM                                                                  (H--L--Lys--Pro).sub.2 Rho 110-4TFA        280-310 10   CD26                   (H--L--Ala--Ala).sub.2 Rho 110-2TFA 4.0  Gly--NaOH  8.7  1 mM DTT 3.6        mM 280-310 10 0.1449  CD26                                                              Ala--Ala                                                                (H--L--Ala--Ala).sub.2 Rho 110-2TFA   8.7 ± .2 8.7  1 mM DTE              280-310 10                                                                       (Z--Ala--Ala).sub.2 Rho 110  Gly--NaOH 7.0-8.5 8.0  1 mM DTE  280-310        10                                                                               (H--L Ala--Ala--Ala--Ala).sub.2 Rho  Gly--NaOH  8.7  1 mM DTE  280-310       10   CD26                                                                        110-2TFA                                                                      TriPeptidyl- (H--L--Ala--Ala--Arg).sub.2 Rho 110-4TFA  Gly--NaOH  8.7          280-310 10                                                                     Pept                                                                           Cathepsin B (H--L--Gln--Ser).sub.2 Rho 110-2TFA 3.6  MES 6.5-7.5 6.5  1       mM DTE 13 mm-22 mM 280-310 10 0.1055 Breast                                             Leupeptin I    Cancer                                                   (H--L--Gln--Ser).sub.2 Rho 110-2TFA  MES 5.5 ± .2 5.5    280-310 10         (H--L--Val--Ser).sub.2 Rho 110-2TFA 4.0  MES 6.5-7.5 6.5   13 mM-22 mM       280-310 10 0.1059 Tumor                                                                 Leupeptin I    Growth                                                          Cystatin C                                                              (H--L--Leu--Gly).sub.2 Rho 110-2Tartrate 1.6-2.0 MES 6.0-6.5 6.5   13        mM-22 mM 280-310 10 0.1037 Progression                                                  Leupeptin I                                                                    Cysteine 2 mM                                                           (H--L--Leu--Gly).sub.2 Rho 110-2Acetate  MES 5.0-6.0 5.5    280-310 10         (H--L--Val--Lys).sub.2 Rho 110-4TFA 1.6  MES  6.5  1 mM DTE 13 mM-22         mM 280-310 10 0.1072 Lung Tumor                                                         Leupeptin I                                                             (H--L Leu--Leu--Arg).sub.2 Rho 110-4TFA 6.4  MES  6.5   13 mM-22 mM          280-310 10 0.1232 Leukemia                                                              Leupeptin I                                                             (H--L--Leu--Gly--Leu--Gly).sub.2 Rho 6.4  MES  6.5   13 mM-22 mM             280-310 10 0.1083                                                                110-2TFA       Leupeptin I                                                     (H--L--Val--Lys--Val--Lys).sub.2 Rho  MES  6.5  1 mM DTE Leupeptin I         280-310 10                                                                       110-6TFA       1 mM EDTA                                                       (H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA        280-310 10                     (H--L--Arg--Arg).sub.2 Rho 110-6TFA       13 mM-22 mM 280-310 10             Gastric                                                                                 Leupeptin    Cancer                                                    Cathepsin B1 (H--L--Leu--Leu--Arg).sub.2 Rho 110-4TFA 6.4  MES 7.0 ±       .5 6.5   13 mM-22 mM 280-310 10 0.1232 Smokers                                          Leupeptin                                                               (H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA  MES  6.5   13 mM-22 mM             280-310 10                                                                              Leupeptin                                                              Cathepsin C (Z--Ala--Gly).sub.2 Rho 110  Gly 7.5 ± .5 7.5    280-310       10                                                                               (H--L--Ala--Gly).sub.2 Rho 110-2Acetate 2.4  Gly 8.0-8.5 8.7                 280-310 10 0.1473                                                                (H--L--Thr--Pro).sub.2 Rho 110-2TFA 2.4  Gly 7.5-9.0 8.7    280-310 10       0.1473                                                                           (Z--Thr--Pro).sub.2 Rho 110   6.0-9.0 7.5    280-310 10                        (H--L--Pro--Arg).sub.2 Rho 110-4TFA  Gly  8.7    280-310 10                   Cathepsin D (H Gly--Leu).sub.2 Rho 110-2TFA 1.2  MES 5.0 ± .5 6.5          10 mm 280-310 10 0.1031 Breast                                                          Pepstatin II    Cancer                                                  (H--L--Thr--Pro).sub.2 Rho 110-2TFA 2.4  MES 5.0 ± .2 6.5                 280-310 10 0.1043 MS Liver                                                                   Disease                                                           Neutral Endo (H Gly--Pro--Leu--Gly--Pro).sub.2 Rho 3.2  MES 7.0 ± .5       6.5    280-310 10 0.1051 Leuk (ALL) CD10                                        Peptidase 110-2TFA                                                              (H Gly--Phe--Gly--Ala).sub.2 Rho 110-2TFA      Zinc Δ  280-310         10   (CALLA)                                                                     (H--L--Arg--Gly--Glu--Ser).sub.2 Rho        280-310 10                         110-4TFA                                                                      EndoPept I (H--L--Arg).sub.2 Rho 110-4TFA  .1M Tris  7.5    280-310 10             Hcl                                                                         (H--L--Glu--Gly--Arg).sub.2 Rho 110-4TFA        280-310 10                    EndoPept II (H--L--Arg--Arg).sub.2 Rho 110-6TFA  PO4  7.0  1 mM DTE           280-310 10                                                                       (H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA      1 mM EDTA  280-310 10              Membrane (H--Gly--Ala--Ala--Ala).sub.2 Rho        280-310 10                        Assoc. 110-2TFA                                                        EndoPept I                                                                     Membrane (H--L--Arg--Arg).sub.2 Rho 110-6TFA        280-310 10                 Assoc (H--L--Ala--Arg--Arg).sub.2 Rho 110-6TFA        280-310 10                      EndoPept II                                                             Glutathione (H--L--Glu--Cys--Gly).sub.2 Rho 110-2TFA        280-310 10          Chymotrypsin (H--L--Glu--Gly--Phe).sub.2 Rho 110-2TFA .1M   7.0   PMSF       280-310 10                                                                        Tris                                                                         Trypsin (H--L--Arg).sub.2 Rho 110-4TFA    7.0   Antipain 280-310 10                 (H Gly--Gly--Arg).sub.2 Rho 110-4TFA       13 mM-22 mM 280-310 10                  Leupeptin                                                             Ester (N-Acetyl MET).sub.2 Rho 110 .1M   6.5    280-310 10                     Proteinase  PO4                                                                γ                                                                       GT (γ-Glu).sub.2 Rho 110-2TFA    7.0  1 mM  280-310 10                           Gly--Gly                                                                Elastase (H--L--Ala--Ala--Tyr).sub.2 Rho       α                        1-Antitrypsin 280-310 10                                                         110-2TFA                                                                       (H--L--Ala--Ala--Pro--Ala).sub.2 Rho        280-310 10                         110-2TFA                                                                       (H--L--Ala--Ala--Ala).sub.2 Rho 110-2TFA        280-310 10                     (H--L--Ala--Pro--Ala).sub.2 Rho 110-2TFA        280-310 10                    Plasmin (H--L--Ala--Phe--Lys).sub.2 Rho 110-4TFA       13 mM-22 mM            280-310 10                                                                              Leupeptin I                                                             (H--L--Glu--Lys--Lys).sub.2 Rho 110-6TFA        280-310 10                     (H--L--Val--Leu--Lys).sub.2 Rho 110-4TFA        280-310 10                    Urokinase (H--Gly--Gly--Arg).sub.2 Rho 110-4TFA        280-310 10                     (H--Gly--Arg).sub.2 Rho 110-4TFA        280-310 10                      HIV Protease (H--L--Lys--Ala--Arg--Val).sub.2 Rho        280-310 10                 110-6TFA                                                                   (H--L--Lys--Ala--Arg--Val--Phe).sub.2 Rho        280-310 10                    110-6TFA                                                                      v-Thrompsin (H--L--Val--Pro--Arg).sub.2 Rho 110-4TFA       PMSF 280-310       10                                                                              Pancreatic (H--L--Pro--Phe--Arg).sub.2 Rho 110-4TFA        280-310 10            Cathepsin L (H--L--Phe--Arg).sub.2 Rho 110-4TFA       13 mM-22 mM           280-310 10  Breast                                                                      Leupeptin    Carcinoma                                                             growth                                                                         cancer                                                             Cathepsin H (H--L--Arg).sub.2 Rho 110-4TFA   8.0 ± .2     280-310 10        Breast                                                                                     Carcinoma                                                          Collagenase (H--Gly--Pro--Leu--Gly--Pro).sub.2 Rho  MES 5.5 ± .2 5.5       MgCl.sup.2 DTE Bestatin 280-310 10                                               110-2TFA                                                                      Neutral FL(palmitate).sub.2 .1 Hanks 7.5     280-310 10 .15                                 Esterase                                                          Acidic Esterase  .1 Mes 6.5     280-310 10 .10                                                           Acid FL(phosphate).sub.2 -2NH.sub.4.sup.+ .2        Mes 5.0     280-310  5 .10                                                                                                     Phosphatase                     Alkaline FL(phosphate).sub.2 -2NH.sub.4.sup.+ .2 Gly 8.7     280-310  5        Phosphatase                                                                    Tartrate FL(phosphate).sub.2 -2NH.sub.4.sup.+ .2 Tartrate 5.2                 280-310  5 .10                                                                   Hairy Cell                                                Resistant          Mes         Leukemia                                                            Phosphatase                                                                    Acid Rho 110(phosphate).sub.2 .1 Mes 5.0     280-310                           5                  Phosphatase                                                 Alkaline Rho 110(phosphate).sub.2 .1 Gly 8.7     280-310                       5              Phosphatase                                                     Tartrate Rho 110(phosphate).sub.2 .1 Tartrate 5.2     280-310  5                      Resistant   Mes                                                         Phosphatase                                                                    Neutral Non- Fluorescein(acetate).sub.2 .1 Hanks 7.5     280-310  3 .15        Monocytes,                                                                     specific            Megakaryo-                                                 Esterase            cytes, Lympho-                                             Acidic Non-  .1 Mes 4.0-6.5     280-310  3 .10                                  cytes                    specific                                             Esterase                                                                       Neutral FL(propionate).sub.2 .1 Hanks 7.5     280-310  3 .15                               Esterase                                                           Acid Esterase  .1 Mes 6.5     280-310  3 .10                                                               Neutral FL(chloroacetate).sub.2 .1 Hanks          7.5     280-310  3 .15                                                           Immature                                          Chloroacetate                  Neutrophils &                                                               Esterase            Mast cells                                                 Acidic  .1 Mes 6.5     280-310  3 .15                                                                             Chloroacetic                                Esterase                                                                       Neutral FL(butyrate).sub.2 .1 Hanks 7.5     280-310  3 .15  Monocytes &        Esterase I            Megakaryo-                                               Acidic            cytes                                                        Esterase I  .1 Mes 6.5     280-310  3 .10                                                                     Neutral FL(chlorobutyrate).sub.2 .1            Hanks 7.5     280-310  3 .15                                                                                                 Esterase I                        Acidic  .1 Mes 6.5     280-310  3 .10                                                                             Esterase I                                  Neutral FL(valerate).sub.2 .1 Hanks 7.5     280-310  3 .15                                   Esterase                                                         Acidic  .1 Mes 6.5     280-310  3 .10                                                                             Esterase                                    Neutral FL(hexanoate).sub.2 .1 Hanks 7.5     280-310  3 .15                                 Esterase                                                          Acidic  .1 Mes 6.5     280-310  3 .10                                                                             Esterase                                    Neutral FL(heptanoate).sub.2 .1 Hanks 7.5     280-310  3 .15                               Esterase                                                           Acidic  .1 Mes 6.5     280-310  3 .10                                                                             Esterase                                    Glycopyrano- (Acetyl-α-D-glucopyranosyl).sub.2 Rho 110  .24 Mes         6.8     280-310 10                                                              sidase                                                                         Glucuronidase (B-D-glucuronide).sub.2 Rho 110  .24 Mes 5.0     280-310        10  Leukemia                                                                    Galactopyrano- (B-D-Galactopyranoside).sub.2 Rho 110  .24 Hanks 7.5           280-310 10                                                                      sidase                                                                         Tyrosine (H--L-Tyrosine Phosphate).sub.2 Rho .1 Hanks 6.5     280-310         5  Cell Cycle                                                                   Phosphtase 110-2TFA           Cell Division                                    Serine (H--L-Serine Phosphate).sub.2 Rho .1 Hanks 6.5     280-310  5          Cell Cycle                                                                      Phosphatase 110-2TFA           Cell Division                                   Threonine (H--L-Threonine Phosphate).sub.2 Rho .1 Hanks 6.5     280-310        5  Cell Cycle                                                                  Phosphatase 110-2TFA           Cell Dividion                                   Neutral (N-Acetyl-L--Ala).sub.2 FL .1 Hanks 7.5     280-310  5                Monocytic                                                                       Esterase II  .1 Mes 6.5     280-310   Leukemias 45 RD                          Adenosine (Adenosine) Rho 110-2TFA .1 Mes 6.0     280-310 10  AIDS                  Deaminase                                                                 Thymidine (Thymidine).sub.2 Rho 110 .1 Mes 6.0     280-310 10                  Deaminase                                                                      Cytosine (Cytosine).sub.2 Rho 110-2TFA .1 Mes 6.0     280-310 10                      Deaminase                                                               Guanine (Guanine).sub.2 Rho 110-2TFA .1 Mes 6.0     280-310 10                 Deaminase                                                                      5'Nucleotidase (Adenosine Menophosphate).sub.2 Rho .1 Hanks 7.5-9.0           280-310 10  Pap Smear                                                            110-2TFA                                                                      Adenine Mono- Rho 110(AMP).sub.2 -4NH.sub.4.sup.+ .1 Mes 6.0                  280-310 10  AIDS                                                                phosphate                                                                      Deaminase                                                                      Angiotensin (Hippuyrl-His--Leu).sub.2 Rho 110 .1 HEPES 8.0     280-310        10  CALLA                                                                       Converting                                                                     Enzyme                                                                         Cholinesterase FL(Choline).sub.2 .1 Hanks 8.0  Zn.sup.++   280-310 10            Cholinesterase FL(Buytryl-Thiocholine).sub.2 .1 Hanks 8.0  Zn.sup.++         280-310 10                                                                     Acetyl FL(Acetyl-Choline).sub.2 .1 Hanks 8.0  Zn.sup.++   280-310 10              Cholinesterase                                                              Nucleosidase (Adenine).sub.2 Rho 110-2TFA .1 Hanks 7.4     280-310 10         Pap Smear                                                                       Lipase (Saturated Hydrocarbon).sub.2 Rho 110 .1                                Lipase (Unsaturated Hydrocarton).sub.2 Rho 110 .1                              Lipase (Triacetin).sub.2 Rho 110 .1 Hanks 7.7                                  Phospholipase Rho 110 (Phosphatidylcholine).sub.2 -2TFA .1 Hanks 7.0           280-310 10 .15                                                                                                                           Phospholipase       Rho 110 (Phosphatidylinositol).sub.2 .1 Hanks 7.0     280-310 10 .15               C                                                                           Phospholipase (Phosphatidyl-choline).sub.2 Rho 110-2TFA .1 Hanks 7.0           280-310 10 .15                                                                                                                           D                    Phospholipase (Phosphatidyl-choline).sub.2 Rho 110-2TFA .1 Hanks 7.0           280-310 10 ..                                                                                                                             A                    (H--L-thyroxine).sub.2 Rho 110-2TFA                                          .sup.1 Range determined experimentally with cells                              .sup.2 pH from scientific literature using cytosol                        

Preferred peptide leaving groups that react with cellular enzymes areincluded in Table 1. As examples, the enzymes glutamyltranspeptidasereacts with gamma-glutamyl amino acid peptide giving gamma glutamicacid; trypsin cleaves the peptide at the arginine residue;aminopeptidase-M hydrolyzes the peptide at the aliphatic amino acidresidue; and chymotrypsin cleaves the peptide at the phenylalanineresidue.

It has been discovered that when the leaving group is a salt complex, itwill significantly improve the transmission of the assay compound intothe cell. The selection of an appropriate salt complex requires aconsideration of the compatibility with the cell, solubility in theaqueous media, and cleavage by the enzyme. Particular care is requiredin the selection of the peptide salt since even isoenzymes have beenfound to be specific in their recognition of particular salts.

Leaving groups for saccharidases are preferably prepared by thesynthesis of monosaccharides, oligosaccharides or polysaccharidescomprising between one and about ten sugar residues of theD-configuration. Examples of useful sugars are monosaccharides-pentoses;ribose; deoxyribose; hexose: glucose, dextrose, galactose;oligosaccharides-sucrose, lactose, maltose and polysaccharides likeglycogen and starch.

The sugar can be an alpha or beta configuration containing from 3 to 7and preferably 5 to 6 carbon atoms. Analogs of these sugars can also besuitable for the invention. Preferably, the D-configuration of themonosaccharide or disaccharide is utilized. The monosaccharide ordisaccharide can be natural or synthetic in origin.

Leaving groups for nucleases, nucleotidases, and nucleosidases arepreferably prepared by the synthesis of nucleic acids, purines,pyrimidines, pentoses sugars (i.e., ribose and deoxyribose) andphosphate ester. Examples are adenine, guanine, cytosine, uracil andthymine. Leaving groups for restriction enzymes would includepolynucleotides.

The nucleic acids contain a purine or pyrimidine attached to a pentosessugar at the 1-carbon to N-9 purine or N-1 pyrimidine. A phosphate esteris attached to the pentose sugar at the 5' position. Analogs of thesebuilding blocks can also be used.

Leaving groups for lipases are preferably prepared by the synthesis ofsimple lipids, compound lipids or derived lipids. Simple lipids can beesters of fatty acids, triglycerides, cholesterol esters and vitamin Aand D esters. Compound lipids can be phospholipids, glycolipids(cerebrosides), sulfolipids, lipoproteins and lipopolysaccharides.Derived lipids can be saturated and unsaturated fatty acids and mono ordiglycerides. Analogs of these lipids can also be used.

Examples of lipids are: triglycerides--triolein, fatty acids--linoleic,linolenic and arachidonic; sterols--testosterone, progesterone,cholesterol; phospholipids--phosphatidic acid, lecithin, cephalin(phosphatidyl ethanolamine) sphingomyleins; glycolipids --cerebosides,gangliosides.

Leaving groups for esterases are preferably prepared by the synthesis ofcarboxylic acids comprising between 2 and 30 carbon atoms. Thecarboxylic acids can be saturated or unsaturated. The carboxylic acidpreferably contains 2 to 24 carbons and more preferably 4 to 24 carbonatoms. Analogs of theses carboxylic acids can also be used. Thecarboxylic acids can be natural or synthetic in origin. Examples arebutyric, caproic, palmitic, stearic, oleic, linoleic and linolenic.

Leaving groups for phosphatases are preferably prepared by the synthesisof phosphates, phosphatidic acids, phospholipids and phosphoproteins.Analogs of these compounds can also be used. Examples are ATP, ADP, AMPand cyclic AMP (c-AMP).

Leaving groups for peptidases are preferably prepared by the synthesisof peptides comprising between one and about ten amino acid residues ofthe L-configuration. Typically, it has been found that the synthesis ofpeptides having more than about six amino acids produces a low yield.However, where the yield is acceptable, peptides of greater length canbe employed.

The amino acids preferably contain 2-10 and preferably 2-8 carbon atoms.Analogs of these amino acids can also be suitable for the invention. Ifthe amino acids are chiral compounds, then they can be present in the D-or L- form or also as a racemate. Preferably, the L- configuration ofthe amino acid is utilized. The amino acids of the oligopeptide can benatural and/or of synthetic origin. Amino acids of natural origin, suchas occur in proteins and peptide antibiotics, are preferred. Syntheticamino acids can also be used, such as pipecolic acid, cyclohexylalanine,phenylglycine, α-aminocyclohexylcarboxylic acid, hexahydrotyrosine,norleucine, or ethionine.

Protecting (Blocking) Groups

Protecting groups are preferably employed when synthesizing the leavinggroup to prevent undesired side reactions of the leaving group duringsynthesis of the assay compound. N-terminal protecting groups and polarorganic protecting groups on the other portion of the amino acidmolecule are used to prevent undesired side reactions of the amino acidsduring syntheses of the peptides. The protecting groups, also known asblocking groups, are removed prior to the assay, unless the presence ofa particular blocking group or groups is found not to interfere with theassay.

The N-terminal protecting groups include an arylcarbonyl, alkylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, arylsulfonyl,alkylsulfonyl, or other equivalents known to those skilled in the art ofpeptide syntheses. The polar organic protective groups include hydroxyl,guanidinyl, sulfhydryl and carboxyl or other equivalents known to thoseskilled in the art of peptide syntheses. Gross and Meienhofer, eds., ThePeptide, 3(3-81) (Academic Press, New York, 1981), describe numeroussuitable amine protecting groups.

Preferred examples of the N-terminal blocking groups include formyl,acetyl, trifluoroacetyl, benzyloxycarbonyl, phthaloyl, benzoyl,acetoacetyl, chloroacetyl, phenoxycarbonyl, carbobenzoxy, substitutedbenzyloxycarbonyl, tertiarybutyloxycarbonyl, isopropyloxycarbonyl,allyloxycarbonyl, phthaloyl, benzoyl, acetoacetyl, chloroacetyl,phenoxycarbonyl, methoxysuccinyl, succinyl, 2,4-dinitrophenol, dansyl,p-methoxybenzenesulfonyl, and phenylthio.

Preparation of Intermediate Complex

A compound containing a blocking group and a leaving group such as anamino acid is reacted with an agent to form an active intermediatecomplex. The leaving group is selected based on the leaving groupdesired in the final assay compound. Suitable agents are known to thoseskilled in the art of peptide chemistry. Examples of suitable agentsinclude carbodiimides, (preferably1-ethyl-3-(3'-dimethylaminopropylcarbodiimide hydrochloride) andbenzotriazolyl-N-oxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) and 1-hydroxybenzotriazole (HOBT reagent). The reagentsare typically stirred in a flask at room temperature. The chemicalstructure of the intermediate complex is presently unknown. The presenceof the complex can be confirmed by thin layer chromatography.

Preparation of Reaction Product

The intermediate complex is further reacted with a compound containingan indicator group (indicator compound) to form a reaction product. Asappreciated by those skilled in the art of peptide chemistry, theindicator compound is dissolved in a solvent to facilitate the reactionwith the intermediate complex. The reagents are typically stirred in aflask at room temperature for a time sufficient to form a reactionproduct. The reaction product can be confirmed by developing a thinlayer chromatography (TLC) plate in an organic solvent. The reactionproduct should be a non-fluorescent compound. When the indicator groupis rhodamine 110, rhodol or a derivative, the presence of the reactionproduct is confirmed by contacting the reaction product with an acidicsolution, such as hydrochloric acid, which cleaves the leaving groupthereby forming a colored product. When the indicator group isfluorescein or a derivative, the presence of the reaction product isconfirmed by contacting the reaction product with a basic solution, suchas sodium hydroxide, which cleaves the leaving group thereby forming acolored product. If only one spot on the TLC plate gives a positive testand there are no trace amounts of fluorescence or its derivatives, thereaction product is of acceptable purity for this stage of the process.

Purification of Reaction Product

The reaction product is then separated from other side reactionproducts, by-products and starting materials in the following manner.Preferably, the reaction product is concentrated to an oil under reducedpressure so as to remove volatile solvents that might be present. Thereaction product oil is then redissolved in a minimum of an organicsolvent, preferably chloroform, methylene chloride, and furtherseparated from the other side reaction products, by-products andstarting materials by normal phase preparative high pressure liquidchromatography (HPLC). Other conventional methods of separation can beemployed. Separation of the reaction product is verified by TLC, aspreviously described, and analytical reverse phase HPLC. The reversephase HPLC will depict the presence of one major band of reactionproduct.

The reaction product is separated from the other side reaction products,by-products and starting materials so that the reaction product can befurther processed by having the blocking groups removed. If the reactionproduct is not sufficiently separated from the other side reactionproducts, by-products and starting materials, then a low yield of theassay compound containing an indicator group and leaving group will beobtained. Moreover, the quality of the separation will have an effect onthe amount of purification that will be subsequently necessary to obtainan assay compound for use in the metabolically active cell.

Removal of Blocking Group

The blocking group which is blocking (protecting) the leaving group isthen removed from the reaction product to obtain an assay compound("intermediate compound" if a salt is to be formed) which contains anindicator group and a leaving group. The reactions are conducted toobtain a free amino acid xanthine derivative by methods known to thoseskilled in the art. When the blocking group on the indicator groupcomprises benzyloxycarbonyl (CBZ), the blocking group is removed by acatalytic reaction of the reaction product in an organic solvent withhydrogen in the presence of palladium or platinum. Further details ofthis process are shown in Example 13. When the blocking group on theindicator group comprises 9-fluorenylmethyloxycarbonyl (FMOC), theblocking group is typically removed by the reaction of the reactionproduct in a polar solvent with an organic base. Further details of thisprocess are shown in Example 1.

To confirm that the blocking group has been removed from the resultingintermediate compound, the intermediate compound is analyzed byanalytical reverse phase HPLC. In addition, the resulting intermediatecompound can be further confirmed by developing a thin layerchromatography plate in an organic solvent.

Physiologically Acceptable Salt Formation

This intermediate compound having an indicator group and leaving groupis then reacted with an acid or a base to form an assay compound, whichis a physiologically acceptable salt. It is important according to themethod of the invention that the assay compounds be physiologicallyacceptable to the cell. The selection of the acid or base has a materialaffect on whether the resulting assay compound will be physiologicallyacceptable to the cell. In addition, it has been found that theselection of the acid affects the selectivity of the assay compound forthe enzyme to be assayed.

It has been found that hydrogen bromide (HBr), even when buffered, killscells. To confirm whether an acid will be appropriate to use, a selectedacid is used to make an assay compound. The assay compound is thentested with a metabolically active cell to determine if viability(Trypan Blue; propidium iodide-fluorescein diacetate PI-FDA!) over theassay time period is affected. Viability is confirmed with Trypan Blueor PI-FDA over a time period of 10 seconds to 30 minutes. If theviability of the cell sample at between one and three million cells/mLdecreases by10% then the salt of the compound is rejected and anothersalt of the assay compound is synthesized.

Preferably the acid that is used to form the salt is selected from thegroup consisting of hydrochloric, sulfuric, nitric, maleic, acetic,trifluoroacetic, tartaric acid, citric, succinic and p-toluenesulfonicacid. More preferably the acid is selected from the group consisting ofacetic, trifluoroacetic, tartaric acid, and p-toluenesulfonic acid. Mostpreferably the acid is trifluoroacetic. When a base is used, ammonia ororganic bases can be used. Most preferably, the base is ammonia.

Purification of the Assay Compound

The assay compound is purified, preferably by reverse phase HPLC. It isvery important that the side reaction products, by-products and startingmaterials from the synthesis of the assay compound be removed whichwould diminish the utility of the assay. Non-physiologically acceptableimpurities should be removed. In addition, the background noisegenerated from impurities should be less than the auto-fluorescence of ametabolically active cell.

It has been found that when a leaving group is present as an impurity,the leaving group can be an inhibitor to enzyme activity. Still further,metal impurities in any of the starting materials can poison theenzymes, prevent hydrolysis of the assay compound and interfere with theaccuracy of the enzyme assay.

In addition, impurities will create background fluorescence which willadd to the natural fluorescence of the cell to create a level ofbackground noise which can interfere with the detection of enzymegenerated fluorescence. Fluorescent impurities can be taken up by thecell, and a rate measurement of fluorescence against time will show afalse rate of increasing fluorescence that is due only to this cellularuptake of fluorescent impurities. This is a particular problem if theassay is conducted to determine the presence or absence of an enzyme,since this impurity will indicate a rate of fluorescence which willfalsely appear to be attributable to enzymatic activity.

The assay compound can be purified by techniques known in the art. Asshown in Example 1, the purification of rhodamine 110 substrate can beaccomplished by reverse phase column chromatography.

In the case of the preparation of salts of peptide-rhodamine 110compounds, a significant level of impurities is created. Theseimpurities include free indicator compound, monosubstituted rhodamine110, blocked amino acids and peptides.

The fluorescence impurities should be removed to a level that they donot obscure the baseline detection of the enzyme in the cell. Thebaseline detection can be established by analyzing log dilutions of anindicator group. Preferably the impurities should be removed so that thefluorescence of the impurities is less than the auto-fluorescence of themetabolically active cell.

Assays for peptidases using assay compounds generate fluorescencegenerally in the range of 10⁻⁵ to 10⁻⁶ Molar free rhodamine 110.Therefore, it is preferred that the free rhodamine 110 and blockedpeptide impurities in the assay reagent should be removed to aconcentration of less than the fluorescence generated by about 1×10⁻⁶ Mand more preferably less than the fluorescence generated by about 10⁻⁷Molar free indicator group. This amounts to a 100,000 photon count usingrhodamine 110 as a standard at 10⁻⁷ -10⁻⁸ M, preferably 5×10⁻⁸ M in a 1cm path length cuvette when measured over 10 min. on a photon countingspectrofluorometer manufactured by the SLM Company of Chicago, Ill. Thiscorresponds to a use level on the flow cytometer where no cellular falsepositive can be detected for a 10 minute period at the highestsensitivity setting. In the case of the peptide-rhodamine 110 compounds,this has been found to require a concentration of impurities of lessthan one part per one hundred thousand, more preferably less than onepart per five hundred thousand, most preferably less than one part permillion. The presence of impurities causes a decrease in the storagestability of the compound, resulting in an increased autohydrolysiswhich leads to increased background fluorescence. A compound should befree of impurities such that when the compound (or reagent containingthe compound) is stored at 4° C. for 30 days, preferably 90 days, morepreferably 180 days, most preferably one year, the backgroundfluorescence increases less than 10%, preferably less than 5%, mostpreferably less than 1% over these time periods, respectively. Thepurified compound or lyophilized reagent are stored in a sealedcontainer over dry nitrogen under atmospheric pressure. The startingpoint in time for measuring stability is usually immediately afterpurification of the assay compound is completed but it can be any timesuch as immediately after the preparation of the assay reagent iscompleted.

Normal phase preparative HPLC procedures are presently preferred toseparate peptide-indicator compound from the impurities. As is known inthe art, solvents of varying polarity can be mixed in varyingconcentrations in order to more effectively separate thepeptide-indicator compound from the various impurities. Thin layerchromatography (TLC) can be utilized to test for the presence of therhodamine 110 substrate in the eluate. This is done by placing a drop ofthe eluent on the TLC plate, and then treating the spot with a suitableacid, such as HCl, to detect the presence of the rhodamine 110substrate, which will turn bright yellow when treated with acid.Analytical reverse phase high pressure liquid chromatography is used totest the peptide-indicator product for purity, as evidenced by a singlesharp band in the absorption spectrum.

Preparation of Assay Reagent

The assay reagent must be compatible with the metabolically active cell.The assay reagent should have an osmolality of from about 250milliosmoles to 350 milliosmoles, preferably from about 275 milliosmolesto 320 milliosmoles. Further, the assay reagent will have an ionicstrength between about 0.1 to 0.3 μ. In addition, the pH of the assayreagent will be between about 4.0 and 9.5, preferably between about 5.0and 8.0. The preferred pH for assay compounds for particular enzymes isincluded in Table 1.

It has been further found that the efficacy of an intracellular assay issubstantially improved by the addition of one or more components in theassay reagent. Examples of improvements include a reduction of reactiontime, increased selectivity for the targeted enzyme, reduction ofcompeting enzyme reactions, increasing signal of enzyme reaction,increasing reactivity of the assayed enzyme relative to othernon-targeted enzymes, increasing the retention time of the indicatorgroup within the cell and other similar advantageous results.

Additional components include buffers, cofactors, modulators,inhibitors, activators for increasing activity of the target enzymesover other non-targeted enzymes, solubilizing components and retentioncomponents can be included in the assay reagent to improve the enzymeassay results. These components are physiologically acceptable to themetabolically active whole cell that is being assayed.

The chemical nature of the buffer is important to the reactivity of theassay compound with the cellular enzymes. For example, it has been foundthat Hanks solution is a better cellular buffer than cacodylic acid at0.1M concentration for amino peptidase. More specifically, by utilizingHanks solution, at pH 7.5, it has been further found that the assaycompound has a higher sensitivity for the targeted enzyme. In addition,the assay compound hydrolysis by the enzyme occurs at an increased rateof reaction. Although Hanks solution contains calcium chloride at aconcentration of 1.26 mM, calcium chloride has been found in the case ofaminopeptidase to be inhibitory to the enzyme reaction with the assaycompound, (H-L-Asp)₂ rhodamine 110, at concentrations of approximately10 mM.

Buffer components that show no inhibitory effect to the cells can beused. Suitable buffer components areN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), Hanksbalanced salt and 2-N-morpholinoethanesulfonic acid (MES), tris-glycine,HEPES, glycine sodium hydroxide, and cacodylate. The preferred buffercomponents are MES for acidic solutions, Hanks for neutral solutions,and glycine sodium hydroxide for basic solutions. Preferred buffers forparticular enzymes are included in Table 1. A metabolic energy sourcesuch as a sugar (glucose) can be added.

Cofactors are components not consumed in the enzymatic reactions, butare required to make the enzyme function. Suitable cofactors includemetals such as Ca⁺², Zn⁺², Mg⁺², Fe⁺² and Mn⁺². These cofactors canincrease the selectivity of the enzyme for the leaving group. Thecofactors can also be co-enzymes or vitamins. Preferred cofactors forparticular enzymes are included in Table 1.

Modulators are components used to decrease the sensitivity of the enzymefor the leaving group. The modulators speed up or slow down the activityof an enzyme by changing the active site. Therefore, enzyme activity canbe down-regulated, as in negative feedback inhibition by the leaving(stimulated) group inhibiting the original enzyme. For example,dithioerythritol (DTE) at 1 mM decreases the sensitivity of thesubstituted rhodamine 110 substrates containing the amino acids Pro,Gly, Gln-Ser, Val-Lys-Val-Lys, Ala-Ala, and Ala-Ala-Ala-Ala, but doesnot change the sensitivity of the leaving group for the enzyme where thesubstrate contains the amino acids Ala-Gly, and Leu-Gly. Dithiothreitol(DTT) has also been found to be an effective modulator. Preferredmodulators for particular enzymes are included in Table 1.

Inhibitors and poisons (or toxins) are components that can be added toreduce the activity of non-targeted enzymes that provide competingreactions for the leaving group. Inhibitors are usually very selectivefor a particular enzyme. For example, EDTA only works with enzymesrequiring Ca⁺², Mg⁺² and some other metals. Other examples of aninhibitor are bestatin, which selectively inhibits aminopeptidase andleupeptin which selectively inhibits cathepsin B. In addition, themonopeptide reagent contains approximately 137.9 mM per liter of sodiumchloride. However, the addition of 7mM of sodium chloride to thedipeptide reagent has a slightly inhibitory effect over the pH range of6.5 to 8.7. Preferred inhibitors for particular enzymes are included inTable 1.

The assay compound must be soluble in the aqueous media. Solubility ismeasured by light scatter using the percent transmittance of light (orabsorbance) through the mixture of the media and assay compound. Asmeasured on a spectrophotometer, the assay compound should have abackground color at a concentration to be used in an assay of less than1000, preferably less than 800, and most preferably less than 500milliabsorbance units at 340 nanometers (25° C.) blanked againstdistilled or deionized water. The assay compound will usually be used ata concentration of 0.5 to 10 mM. A useful concentration for determiningsolubility is 5mM.

Preferably, a two fold excess quantity of the assay compound that willreact with the enzyme during the time of the assay must be soluble inthe aqueous media. An excess of assay compound is preferred. If aninsufficient amount of the assay compound is provided, the enzymereaction will completely hydrolyze the assay compound and the dynamicrange of the assay will be limited. The resulting indicator compoundwill have a limited fluorescence duration. However, when an excess ofthe assay compound is employed, the enzyme reaction will continuouslyhydrotyze the assay compound and the fluorescence duration will continueduring the enzyme reaction. This provides the advantage of having alonger time period in which to sense for one or more reaction states ofthe assay compound.

A solubilizing agent can be utilized with assay compounds for whichsalts are not available, or where such solubilizing agents will assistthe transfer of the assay compound into a metabolically active cell. Thesolubilizing component is present in an amount effective to enable theassay compound to pass through the cell lipid bilayer withoutdetrimentally affecting the cell. The solubilizing agent should becarefully chosen because the wrong solubilizing agent can cause lysis orcell death.

When the assay compound has a background color (at the concentration tobe used in an assay) greater than 1,000, greater than 800 or greaterthan 500 milliabsorbance units, a solubility component may be used tolower the background color to less than 1,000, less than 800 or lessthan 500 milliabsorbance units. However, the concentration of thesolubilizing component is limited. If a high concentration of thesolubilizing component is used, metabolically active cells will belysed. If a low concentration of the solubilizing component is used,sufficient solubility of the assay compound will not be attained. Theeffective amount of solubilizing component may be empiricallydetermined, but is typically less than 10.0% by weight of the assaycompound.

Suitable solubilizing components include non-ionic surfactants,polyethylene glycol, dimethyl sulfoxide (DMSO), and mannitol, as notedin Table 2. BRIJ 35 and TWEEN 20 are the tradenames for products fromICI Americas, Inc. PLURONIC 25 R8 is the tradename for a product fromBASF Wyandotte. TRITON X100 is the tradename for a product from Rohm andHaas Company.

                  TABLE 2                                                         ______________________________________                                                    COMMERCIAL                                                        CONCENTRATION                                                                             NAME        CHEMICAL STRUCTURE                                    ______________________________________                                        0.1%        BRIJ 35     Polyoxyethylene lauryl                                            (nonionic)  ether                                                 0.2%        PLURONIC    Ethylene oxide with                                               25 R8       hydrophobic base from                                             (non-ionic) propylene oxide and                                                           propylene glycol                                      0.1%        TRITON X100 Octylphenoxy polyethoxy                                           (non-ionic) ethanol                                               0.1%        TWEEN 20    Polyoxyethylene sorbitan                                          (non-ionic) monolaurate                                                                   (polysorbate 20)                                      0.1%                    Polyethylene Glycol                                     5%                    Dimethyl Sulfoxide                                    4.5%                    Mannitol                                              ______________________________________                                    

When using a solubilizing component, certain difficulties have beenencountered. While the solubilizing component facilitates thetransmission of the assay compound into the metabolically active cell,the solubilizing component will also facilitate the expulsion of thefluorescent indicator group compound from the metabolically active cell.The expulsion of the indicator group will have the negative effect ofpermitting non-enzyme containing cells to absorb free dye. When thisoccurs, the accuracy of an enzyme assay is compromised.

In addition, the electronic configuration and polar nature of theliberated indictor dye influences its ability to be retained within thecell. Retention of the dye is important for proper detection.

A feature of the present invention used to avert the problem of cellularexpulsion when using a solubilizing component, is for the assay reagentto include a retention component. The retention component will compriseat least one agent that will inhibit a cell pump mechanism forexpressing extracellular material. Such cell pumps include the multipledrug response pump, calcium channel pump, sodium pump, potassium pumpand ATPase pump. Suitable retention components includetrifluoperazine•HCl, prochlorperazine•emaleate, and chlorpromazine•HClto inhibit the multiple drug response pump; verapamil•hydrochloride toinhibit the calcium channel pump; and digoxin (C₄₁ H₆₄ O₁₄), digoxinderivatives, such as ouabain (C₂₉ H₄₄ O₁₂), and strophatidin (C₂₃ H₃₂O₆) to inhibit the sodium, potassium and ATPase pump.

The media in which the assay compound is dissolved must be compatiblewith the cell so that the cell can remain metabolically active in themedia for at least the duration of the assay. The media is preferablysterile and free of endotoxin and chemicals that adversely affect thephysiology of the cell. The assay compound is preferably completelysoluble in the media at the concentration at which it is used. The assaycompound is preferably used in concentrations up to the saturation orthe suspension level or before turbidity occurs. The media may bephysiological saline or a buffered solution (phosphate buffered Saline)in which the assay compound and other additives are dissolved. The mediashould preferably include a buffer agent so that the pH of the assaymixture of metabolically active cells and assay compound is maintainedat a point that is appropriate for the enzyme hydrolysis.

For storage purposes the compound and media mixture should belyophilized under conditions where sublimation of the solvent occursupon application of a vacuum. Applying a vacuum to the sample at atemperature where a liquid forms on the solid before going to a gasphase, referred to as "melt back" may cause degradation of the compound.Appropriate temperatures should be determined for each compound, andpreferred temperatures are usually -5° C. to -35° C. for predominantlyaqueous solutions. During the thermal cycle of lyophilization, heat maybe applied after sublimation to drive off any additional moisture. Theproduct temperature should never exceed the heat applied and the productshould be brought to room temperature over 15 to 72 hours. The vacuumshould be returned to atmospheric conditions by bleeding in drynitrogen. The product is stoppered at atmospheric pressure andtemperature. The lyophilized compound is stored at 4° C. to 8° C. andmay be reconstituted using endotoxin-free deionized water.

Auto-hydrolysis, which is the nonspecific hydrolysis of the substrate,yields cellular fluorescence not derived from the target enzyme.Stability of the substrate compound has been demonstrated to be a keyfactor in preventing auto-hydrolysis.

The assay compound and/or the assay reagent should be sufficientlystable so that no auto-fluorescence or chemiluminescence is created bythe degradation of the assay compound prior to cleavage by the enzyme.Preferably, when the assay compound or assay reagent is stored at 20° C.for 30 days, preferably 90 days, more preferably 180 days and mostpreferably one year, the reagent exhibits a photon count of 100,000 orless. Photons can be measured by using a 2 millimolar solution of assaycompound in deionized water and a path length of 1 cm against arhodamine 110 standard as previously described. Fluorescent impuritiesshould account for less than 10% of the fluorescence generated duringthe assay.

An acceptable reagent should have the following three characteristics;(1) there should be a low level of native free fluorescence that isabsorbed by the cells, non-specifically. Thus, there should be a lowlevel of fluorescent impurities such as free indicator compounds. Theacceptable and preferred levels of these impurities have already beendescribed. (2) The reagent should be stable over time so that it doesnot need to be used shortly after it is prepared. Certain impurities andcertain reagent additives can increase the rate of autohydrolysis whichincreases the fluorescence of the reagent. Acceptable and preferredstabilities have already been discussed. (3) The reagent should alsohave a high enough rate of reaction with the enzyme being measured sothat fluorescence generated as a result of reaction between the enzymeand the reagent can be easily measured. In one aspect, the reaction rateshould be sufficiently high that fluorescence generated as a result ofcleavage of the leaving group inside the cell is at least 2 times,preferably at least 10 times, more preferably at least 50 times and mostpreferably at least 100 times greater than other non-specificfluorescence generated in the assay. In another aspect, the reagentshould contain an unblocked assay compound which has a reactionrate-which is at least 2 times, preferably at least 5 times, morepreferably at least 100 times, most preferably at least 1000 times thereaction rate of a corresponding blocked assay compound. For example,the unblocked assay compounds of the present invention which containunblocked amino and or peptide leaving groups have an enzymatic reactionrate which is considerably greater than the reaction rate of thecorresponding compound wherein the amine group(s) on the leaving groupis blocked by, for example, a Cbz group.

Types of Assays

It has been discovered that the assay reagent can be used to determineenzymatic activity of metabolically active whole cells to provideindication of the presence of a disease, of the progress of a disease,the efficacy of a drug, and cell differentiation.

It has been found that the activity of one or more enzymes changes withdisease progression. Changes in the activity of one or more enzymes canbe examined to provide an indication of the presence and progress of adisease. In addition, the measurement of the activity of certain enzymescan provide an indication of the response to certain drugs ortreatments, since the activity of one or more enzymes will change if thedrug is successfully fighting, modulating or treating the disease. Stillfurther, it has been determined that differentiation of a cell can bedetermined by the presence of one or more selected enzymes.

Existing tests for the presence of a disease, progress of a disease, orefficacy of a drug require significant extracellular concentrations ofthe enzymes that are being measured. Usually, hours or days are requiredto allow extracellular concentrations to rise to detectable levels. Thepresent invention has the further advantage of providing a method toproduce a reagent for measuring the intracellular concentrations ofenzymes. This enables the diagnostic assay to obtain analysis of theenzyme of interest in a shorter period of time and to monitorintracellular events as they are occurring.

Enzymatic assays are performed by contacting metabolically active cellswith an assay reagent. The leaving group is selected to be one which canbe cleaved from the indicator group by the targeted enzyme. The reactionoccurs for a period of time sufficient for the leaving group to becleaved from the indicator group by the targeted enzyme. Sensing for oneor more reaction states confirms cleavage of the indicator group by theenzyme.

In some instances, one can characterize a disease, the progress of adisease, or efficacy of a treatment by sensing for only a single enzyme,as in the case where a disease is characterized by the presence or lackof activity of an enzyme. For instance, Hereditary Non-SpherocyticHemolytic Anemia (HNSHA) is distinguished from hereditary spherocytosisby the fact that red blood cells are morphologically normal and manifesta normal osmotic fragility. Only in the case Of pyrimidine5'nucleotidase deficiency is the erythrocyte morphology changed to abasophilic stippling. (See Example 27) However, in most instances theseconditions will be characterized by assaying for the activity of atleast two enzymes. In practice, five or more enzymes will be used in apanel to serve as checks and to reduce the probability of false positiveor false negatives since the activity of a targeted enzyme can bepresent in two different diseases. However, as the number of targetedenzymes are increased in the panel, the assay becomes more reliable ordifferentiable. The detection is more reliable because two differentdiseases will have different enzyme patterns.

Thus, there are at least two ways to run the assay of the presentinvention:

running a single assay and detecting a difference between the beginningstate and the end state of a substrate, such as the cleavage of a singlesubstrate by a target enzyme to yield free peptide and fluorescentindicator dye; and

running a series of assays with a pattern matrix of several substratesreacted with an abnormal cell versus the same matrix reacted with anormal cell.

"Reliability" refers to the ability to make pattern matrix decisionswithout failure. Error in a single test may not, in fact, invalidate apattern matrix. For a small set of assays, the assay provides anincreased capability to differentiate states of abnormality.

The panel of selected enzymes are created by developing a range ofnormal values for enzymatic activities and ratios of enzymaticactivities to each other. This panel will be used to compare the testresults from the cell analyte. The enzyme activities from the examinedcell analyte is compared to at least one of a reference/non-diseasedcell or a reference/diseased cell to obtain an indication of a diseasedstate.

In other instances, the analysis of cellular enzymes involving classesof enzymes provides the ability to sort cells by type or morphology. Asmany as a thousand different enzymes may be operative in any given cell,but only a few dozen enzymes define the unique function or functions ofany one cell type. Many enzymes are inhibited or missing fromfunctionally different cells. Determination of proteases, glycosidases,glucosidases, carbohydrases, phosphodiesterases, sulfatases,thioesterases, pyrophosphatases, nucleotidases, nucleosidases,saccharidases, esterases, phosphatases, lipases and combinations thereofprovides a matrix to rank cells by their functional activity.

Classification of normal cells morphologically can be made bydetermining key enzyme activities. For example, nucleated red bloodcells (NRBC's) can be distinguished from non-nucleated red blood cells(RBC's) by determining dipeptidyl peptidase IV activity in the cellanalyte. In NRBC's dipeptidyl peptidase IV activity will be present butin RBC's dipeptidyl peptidase IV activity will be absent. In addition,the age of RBC's can be determined by the presence or absence ofadenosine deaminase or 5'nucleotidase.

In still other instances, the analysis of cellular enzymes involvingclasses of enzymes provides the ability to study cell proliferation.Cell proliferation is stimulated by growth factors. Cell proliferationis the ability of cells to divide and increase their numbers. Phases ofcell division are under gene control and take a specified time periodfor each part of the division process. The time from one division to thenext includes a randomly variable component. Different cell typesrequire different growth factors in order to divide. All cells competefor growth factors. Cells are programmed for a certain number ofdivisions and then they die.

Abnormal cells that disobey normal constraints on cell divisionproliferate to form tumors in the body. They also appear transformed incell culture. Cell transformation is often accompanied by mutation orover-expression of specific oncogenes.

Some normal cells proliferate as part of their function. Signalingmolecules are produced in the course of the inflammatory response andstimulate the bone marrow to produce more leukocytes. This regulationtends to be cell type specific. More specifically, some bacterialinfections cause a selective increase in neutrophils, while infectionsfrom parasites cause a selective increase in eosinophils.

A blood cell differential can be constructed using this invention todetermine cell types, immature cells, mature cells, abnormal cells dueto drug interreaction and abnormal cells due to disease. For example,cell types can be identified by the assay of the present invention. Forexample, lymphocytes can be distinguished from monocytes or neutrophilsby peroxidase activity. Lymphocytes will not show peroxidase activitywhile neutrophils will show peroxidase activity, and lymphocytes willnot show esterase activity while neutrophils will show esteraseactivity. In addition, acetate esterase activity is present in monocytesthat have been inhibited with sodium fluoride, but absent inneutrophils.

In addition, the analysis of cellular enzymes involving classes ofenzymes provides the ability to study cell activation. Activation of Tcells is a complex process involving various secreted proteins calledinterleukins which act as chemical mediators. Activation is thought tobegin when the T cell. stimulates the antigen presenting cell to secreteone or more interleukins. IL-1 mediator causes the T cell to stimulateits own proliferation by inducing it to secrete a growth factor IL-2, asWell as synthesize IL-2 receptor to initiate proliferation.

Helper T cells are essential for B cell antibody response. Onceactivated by foreign antigen, the T cell presents the antigen to a Bcell for antibody synthesis. Other Helper T cells secrete γ interferonwhich attracts macrophages and activates them to defend againstinfection by microorganisms. Diagnosis of infection from inflammationand inflammatory diseases has been achieved using the assay reagent in apyrogen-free, sterile environment. Activation and proliferation agents,like phorbol myristate acetate (PMA), f-Met-Leu-Phe, IL-1, IL-2, GMCSFand γ interferon are added to the media and specific cell types areobserved for response. Treatment regimes can also be monitored foreffectiveness by using the assay reagent in conjunction with growthstimulators or signal peptides.

The present invention has potential use in the following clinidalapplications: diagnosis of cervical cancer, diagnosis of viralreplication in HIV patients, diagnosis of HIV infected blood in bloodsupply, diagnosis of TB infected HIV patients, improved blooddifferential, differential diagnosis of vital from bacterial infections,differential diagnosis of Lupus from rheumatoid arthritis, differentialdiagnosis between rheumatoid arthritis from osteo arthritis, diagnosisof vasculitis, diagnosis of cardiovascular disease, monitoring ofchemotherapeutic efficacy, diagnosis of Hodgkins Disease, confirmationof gene implantation and diagnosis of transplant rejection.

For a diagnosis of cervical cancer, several enzymes related to thepresence of cervical cancer can be measured.

For a diagnosis of viral replication in HIV. patients, HIV replicationin blood cells can be monitored. A sensitive measure of HIV replicationcan be important as a predictor of rapid movement into the AIDS statefrom the HIV infected stage of the disease. Since the virus replicatesin the lymphocytes and monocytes, monitoring specific enzyme levels canmake the monitoring both inexpensive and reproducible.

Identification of infected units in blood supply is one of the majorgoals of those responsible for the quality of blood supplied fortransfusion to reduce the probability of HIV or Hepatitis infection. Alow cost screening methodology can be devised whereby the blood can besubjected to HIV antibody testing and testing by the method of thisinvention.

In the management of AIDS patients, the early diagnosis of Tuberculosisis important to insure rapid recovery and to reduce the chance offurther complications. The objective of such a test using this inventionis to distinguish TB⁺ HIV patients from TB⁻ HIV patients. The earlyidentification of the TB⁺ HIV patients can permit administration oftherapy to prevent additional complications in these immune deficientpatients.

This invention also has utility for the differential diagnosis of viralfrom bacterial infections. Many patients have an elevated temperatureand it is not known whether the temperature is from a viral or bacterialorigin. The differential diagnosis between viral and bacterialinfections assists the clinician in the management of these patients byallowing the physician to apply the proper therapy on an as neededbasis.

This invention has further utility for differential diagnosis of Lupusfrom rheumatoid arthritis/drug monitoring in rheumatoid arthritis andLupus patients. In the early course of disease, the symptoms for LupusErythematosis and rheumatoid arthritis are sufficiently similar thatdifferential diagnosis of the disease is difficult, especially when aLupus patient has early arthritic involvement. This has clinicalconsequences since it delays the administration of the correct therapy.Lupus can be a clinically aggressive disease and it is beneficial to thepatient to have the correct diagnosis at an early date. These patientshave different enzymes in activated states meaning that this methodologyis the modality to use for a differential diagnosis. Additionally,monitoring the therapeutic application of steroid drugs can be ofbenefit to the patient.

This invention has still further utility for differential diagnosisbetween rheumatoid arthritis from osteo arthritis. Rheumatoid arthritisis an aggressive autoimmune disease which results in destruction of thepanus of the joint. Osteo arthritis is a degenerative disease of theaging joint which is not immune mediated. Since immune cells migratethroughout the body, this methodology provides an early differentialdiagnosis between these two diseases. This is important since thecorrect therapy for each disease is different.

Moreover, this invention has utility for diagnosis of vasculitis.Vasculitis is an autoimmune disease of blood vessels generally in theextremities. Patients with this disease typically have nondescriptcomplaints of pain which do not permit diagnosis until considerabledamage has been completed on the vascular system by the immune cells.Since it is an autoimmune disease caused by circulating immune cells,the disclosed methodology can provide the needed information to make anearly diagnosis.

Furthermore, this invention has utility for monitoring of cardiovasculardisease. Atherosclerosis results in the deposition of platelets andother cellular components into the walls of coronary vessels. Thisprocess results in the loss of elasticity of the vessels and eventuallyin death. It has been shown that in these patients, as many as 20% ofthe platelets are in the activated state. Evaluation of platelets canpermit the identification of patients with active atheroscleroticprocesses ongoing and permit administration of disease altering drugs.

Moreover, this invention has utility for monitoring of chemotherapeuticefficacy. Patients undergoing chemotherapeutic therapy have alteredenzyme patterns which indicates that this change in enzyme levels can beused to monitor the effectiveness of chemotherapy.

In addition, this invention has utility for diagnosis of Hodgkinsdisease. The practice of this invention can be useful to monitor thestages of Hodgkins disease.

Furthermore, this invention has utility for diagnosis of transplantrejection. The practice of this invention can be useful to monitor theacceptance of an organ transplant. All patients are givenimmunosuppressants to prevent organ rejection and therefore it isdifficult to distinguish infection from rejection.

Moreover, this invention has utility for monitoring for metastaticinvasion. It has been found that tumor cells have different patterns ofenzymes from normal cells in the same tissue. Identification of thetypes of enzymes is useful and important for predicting metastaticpotential and invasion. Tumor cells in circulating blood can be usefulto predict the progression of the disease.

Preparation of Metabolically Active Whole Cells

The assay reagent is reacted with a metabolically active whole cellanalyte. The metabolically active whole cells are contained in tissue,blood, cell cultures or other cells containing constituents. Preferably,the metabolically active whole cells are separated into cell types. Themetabolically active cells to be analyzed are isolated by knowntechniques such as differential lysis, differential centrifugation, andaffinity columns. However, separation of the cells to be studied fromother cells is not always essential.

The cells are usually washed to remove any extracellular enzymes,optionally with lysis or physical separation of unwanted cells. Severalpreferred techniques for accomplishing this are summarized in FIGS.1A-1D.

The analysis of the segregated metabolically active cells providesspecificity for a particular enzyme analysis. For example, when themetabolically active cell is a leukocyte blood cell, the methodcomprises separating the leukocyte cell from the cell analyte, washingthe remaining leukocyte cell to remove any serum or plasma enzymes,contacting an assay reagent compound with the leukocyte cell, anddetermining fluorescence from the leukocyte cell (See FIG. 1B). Amodification of this method comprises washing the cell analyte to removeany serum or plasma enzymes, contacting an assay compound with the cellanalyte, separating the leukocyte blood cells from the cell analyte, anddetermining fluorescence from the leukocyte cells (See FIG. 1A). Inaddition, another method that can be used for cell analytes of leukocyteblood cells, nucleated erythrocyte blood cells and platelets analytescomprises washing the cell analyte to remove any serum or plasmaenzymes, contacting an assay compound with the analyte and determiningfluorescence from the analyte (See FIG. 1C).

To confirm that cells are metabolically active at the time of the assay,it is desirable that the viability of the cells be checked at the timeof the assay. Several tests are useful to determine the viability ofcells. Trypan blue is a blue stain which diffuses into the cell and isremoved by cells if the cells are viable. Dead cells will not remove thedye and will take on a blue color. Propidium iodide is a DNA-RNA stainwhich, if the cell is dead and membranes are damaged, will penetrate thecell and stain the DNA-RNA. Fluorescein diacetate-propidium iodide willcause living cells to take on a green color because the fluoresceindiacetate will be hydrolyzed, while dead cells become red from thepropidium iodide. Red blood cells do not undergo cell division, andtherefore a test for the presence of 2,3-diphosphoglucose dehydrogenase(which is an indicator of cell division) is a useful test for viability.

The assay of the present invention is particularly useful for measuringintracellular concentrations of enzymes in mammalian cells such as humancells. However, the assay should also be useful in various or othertypes of cells which have metabolic activity.

Assay Conditions

The concentration of the cells in the media should be high enough toprovide a reading of the desired number of cells within the desired timeperiod, taking into consideration the speed of the instrument that isbeing used. For current flow cytometry techniques, a concentration ofabout three million cells per milliliter is appropriate to yield ameasurement of about 10,000-15,000 cells in about 1-2 minutes.

The assay compound is generally employed in concentrations in excess ofthe amount which can be completely hydrolyzed by the quantity of enzymewithin the time of the assay. An assay compound concentration that istoo high may have a negative affect on enzyme activity, since theleaving group can be a negative feedback inhibitor to enzyme activity.

The leaving group concentration in a cellular optimization is determinedusing Km (a known rate constant) and VMA_(X) (maximum velocity)calculations. The leaving group is preferably present in an amount fromabout 2 to about 100 x V_(MAX) and most preferably from about 2 to about10 times the amount which can be completely hydrolyzed by the enzymewithin the duration of the assay period. Preferred leaving groupconcentrations for particular enzymes are included in Table 1.

The assay may be conducted either as a rate determination or as an endpoint determination. Rate determinations are preferred, because they aregenerally less affected by auto-fluorescence. Consequently, a ratedetermination assay is more sensitive and precise. In a ratedetermination, the fluorescence of the assay compound-cell analytemixture may be determined promptly after the cell analyte is contactedwith the assay compound. The ability to see a signal and distinguish itfrom background noise determines the initial starting point of datacollection and the final data point is preferably determined at thepoint where the slope of the reaction rate changes, typically more than2%.

Most cellular reactions do not strictly obey zero-order kinetics. Mostcellular enzymes show a delay between the time of exposure of the cellsto the assay compound, and the ability to detect a signal that isgreater than the background noise. Cellular enzymatic reactions that donot obey zero order kinetics are still useful measurements as firstorder, pseudo first order, or initial rate measurements. Multipleenzymes in a reaction (mixed reactions) are displayed by slope changesduring the time course being monitored.

In an endpoint determination, the enzyme hydrolysis reaction is allowedto proceed for a predetermined length of time, usually at V_(MAX). Thereaction time can be calculated based on whether the reaction is zeroorder or first order kinetics using Michaelis--Menton methodology.Alternatively, the reaction time can also be adjusted by a differentelapsed time for pseudo-first order reactions.

It has been determined that a number of factors will decrease thereliability of the assay, and yield false positive, or erroneousindications of enzymatic activity. These include (i) extended reactionbetween the cell analyte and the assay compound; (ii) another,non-targeted enzyme that is cleaving the leaving group; (iii)auto-hydrolysis of the assay compound; (iv) inhibitors or stimulatorsthat are present and undetected; (v) cells that are no longermetabolically active, or dead; (vi) mixed populations of cells; (vii) atransfusion of the patient before sampling; (viii) non-specific dyeuptake by negative cells; and (ix) background fluorescence. The creationof false negatives, or false indications of a lack of enzymaticactivity, can be caused by (i) insufficient reaction between the cellanalyte and the assay compound, (ii) a hypoosmotic media leading to adecrease in cell activity; (iii) a cell that is no longer metabolicallyactive; (iv) burst cells; and (v) the presence of inhibitors to thetarget enzyme.

It has been further determined that assays will be significantlyimproved if reaction conditions are adjusted to maximize the activity ofthe assayed enzyme relative to other non-assayed enzymes which mightotherwise compete for the leaving group. More specifically, the targetedenzyme can be involved in a chain cascade reaction of enzymessequentially coupled to other enzymes, as in a multi-enzyme reactioncascade.

The reaction conditions can be adjusted to maximize the efficiency ofthe pathway, or to decrease the efficiency of competing pathways. Suchconditions preferably include at least one of pH, choice of form ofassay compound, temperature, osmotic pressure, ionic strength, andreaction time.

The pH at which an enzyme is most efficient can be determined from theliterature, or determined empirically. As shown by FIG. 2, pH maxima canhave two peaks (optima). Therefore, the selection of the appropriate pHshould be made with care. In addition, care must be exercised when usingpH information from the literature, because these values will usually bebased on cytosol studies and not on intact, metabolically active wholecells. Therefore, it is preferable to use values from the literatureonly as a starting point, and then to determine the appropriate pH fromthis reference point. Generally, the pH will be between about 4.0 and9.5. The pH of the assay mixture is controlled by dissolving the cellanalyte and assay compound in an appropriate buffer. A list of preferredpH's for particular enzymes is included in Table 1.

The form of assay compound can be important since some enzymes requirenon-derivatized, natural structures for recognition of binding andreaction, whereas other enzymes are less selective. More specifically,derivatization and salt formation of the assay compound are importantproperties for solubilization, enzyme recognition and protection fromauto-hydrolysis.

A reaction run using the same data collection window without the enzymesource will determine auto-hydrolysis of the substrate and therefore thepotential for negative cells to absorb the dye non-specificallyresulting in false positive.

The time of the assay is typically less than 30 minutes, preferably lessthan 20 minutes, usually between 5 seconds and 20 minutes, and mostpreferably between about 10 seconds and about 5 minutes. Some enzymesystems, such as esterase and phosphatase, can react with the assaycompound in shorter periods of time due to concentrations of enzymesfound in the cell. The reaction time should be limited so that theeffects of cellular expulsion of the indicator compound will be avoided.Preferred time periods for assaying particular enzymes are included inTable 1.

The temperature at which the assay is performed must be physiologicallyacceptable to the cell. The temperature must be high enough to retainviability and to ensure enzyme activity, but not so high as to causedegradation or other deleterious reactions involving the leaving group,the enzyme, or other components of the mixture. Particular enzymes, orenzymes in particular pathways, are more reactive at particulartemperatures. The temperature is preferably maintained between about 30°C. to about 40° C., more preferably between about 35° C. and about 38°C., and most preferably between about 36° C. to about 38° C. Preferredtemperatures for a variety of enzymes are shown in Table 1.

The osmotic pressure of the assay mixture is controlled to be withinphysiological ranges from about 250 milliosmoles to 350 milliosmoles,preferably from about 275 milliosmoles to 320 milliosmoles. The osmoticpressure must be selected to maintain the viability of the metabolicallyactive whole cell. Variations in osmotic pressures will result in lysisof the cell, severe shrinking or shriveling (crenation) when too low,and swelling or bursting (stomatolysing) of the cell when too high.

The ionic strength of the assay mixture should be selected so as toavoid shriveling crenating or bursting (stromatolysing) of the cells,and also to maximize the activity of the assayed enzyme relative toother, non-assayed enzymes. An ionic strength that is too low coulddeplete metals such as Ca⁺², Mg⁺², and Zn⁺², or cause insufficientamounts of anions such as Cl⁻¹, NO₃ ⁻¹, SO₄ ⁻² and PO₄ ⁻³ which are thecofactors that can be used to improve enzymatic activity. The ionicstrength of the assay reagent is preferably between about 0.1 to 0.3 μ.A list of preferred ionic strength values for particular enzymes isincluded in Table 1.

The fluorescence reading is made after the reaction has occurred orafter a specific period of time. Typically, the reaction is stopped byimmersing the reaction container in ice and water which cools the cellsto about 0° C. Sensing for one or more reaction states by fluorescencedeterminations confirms cleavage of the indicator group by the enzyme.

The fluorescence determinations are performed on a Image Analysis System(IAS) or a Flow Cytometer (FC). The IAS is a microscope based systemthat measures fluorescence known to those skilled in the art. Arepresentative example of an IAS is the Metamorph™ by Universal ImagingCorporation, West Chester, Pa. The structure and operation of flowcytometers is also well documented in the literature. Alternatives totraditional FC include slit-scan FC and stopped-flow FC. The type ofinstrument used to conduct the experiments described in the examples wasa flow cytometer (for example, a Coulter Profile® flow cytometermanufactured by Coulter Corporation of Miami, Fla). This flow cytometermeasures fluorescence across the entire cell. Flow cytometric methodswhich measure fluorescence in only a part of the cell, such as slit scanflow cytometry, have significant utility in the invention because thebackground fluorescence is significantly reduced when measurements arefocused on the region of the cell where the enzyme is located.

The fluorescence determinations can also be taken by aspectrofluorometer which has the capability to measure the very lowfluorescence levels that are generated by the assay. Thespectrofluorometer is tuned to the excitation and emission wavelengthsof the particular indicator being used. Preferred compounds such asrhodamine 110 and fluorescein have excitation and emission wavelengthsof about 495 to 498 nm (excitation) and 520 to 525 nm, respectively. TheModel 8000C photon counting spectrofluorometer manufactured by the SLMcompany, a subsidiary of Milton Roy (Chicago, Ill.) was used.

The flow cytometer can perform additional measurements in addition to asingle wavelength fluorescence measurement. The flow cytometers can beequipped to measure fluorescence at two or more separate wavelengths.Such readings are useful to perform assays according to the inventionwhen using more than one assay compound, or for using cell surfacemarkers, such as monoclonal antibodies, to determine cell morphology.Additional wavelengths are useful to measure the activity of anotherenzyme, which can be a peptidase or a different enzyme such as aphosphatase, saccharidase, nucleotidase, esterase, or lipidase. Suchadditional tests are useful for simultaneously characterizing diseasestates, and for determining cell morphology and cell types.

Assay Protocols

Preferred sample preparations by which enzymes can be assayed using thereagents prepared according to the method of the invention have beendeveloped. These sample preparations can be modified, and are includedherein to disclose those procedures that are currently preferred.

The practice of the cell probe assay is divided into three parts: 1.Sample preparation, 2. Data Collection (i.e., Detection of Fluorescence)and 3. Results (i.e., Data Analysis).

1. Sample Preparation:

Sample preparation can be divided into four different processes A, B, Cand D which are illustrated in FIGS. 1A, 1B, 1C and 1D, respectively.The choice of sample preparation is dependent upon the user and theanalyte. The four processes are:

Process A: Examination of leukocytes or tissue cells with erythrocytecontamination with post-lysing.

A sample, consisting of whole blood (in EDTA, Heparin or ACD) ordissociated tissue or body fluids (synovial fluid) or cell culture mediais obtained and stored in a manner so as not to decrease viability. Thesample is washed sufficiently to remove plasma, media, body fluid,debris and extra-cellular enzymes. The wash media consists of aphysiologically balanced buffered salt solution. The washed cells areincubated at 37° C. 50 μL of sample and 25 μL of substrate media aremixed together and allowed to incubate at 37° C. for a predeterminedamount of time. At the end of the incubation period, unwanted cells arelysed with a lytic reagent, i.e., erythrocytes are removed. Compatiblelytic systems are Q-Prep™, an acid lyse (formic acid/quench),Erythrolyse™, (acid lyse/detergent/quench) or hypotonic ammoniumchloride. The sample is then measured for fluorescence. The referencedlytic systems are commercially available from Coulter Corporation,Miami, Fla.

Process B: Examination of leukocytes or tissue cells with erythrocytecontamination with pre-lysing.

A sample, consisting of whole blood (in EDTA, Heparin or ACD) ordissociated tissue or body fluids (synovial fluid) or cell culture mediais obtained and stored in a manner so as not to decrease viability.Unwanted cells, i.e. erythrocytes, are lysed with a lyric reagent.Compatible lyric systems are acid lysed (formic acid/quench), IVCS lyse(quaternary ammonium salts)/quench or hypotonic ammonium chloride. Thesample is washed sufficiently to remove plasma, media, body fluid,debris and extra-cellular enzymes. The wash media consists of aphysiologically balanced buffered salt solution. The washed cells areincubated at 37° C. 50 μL of sample and 25 μL of substrate media aremixed together and allowed to incubate at 37° C. for a predeterminedamount of time. At the end of the incubation period, the sample is thenmeasured for fluorescence.

Process C: Examination of platelets, erythrocytes, leukocytes,dissociated tissue, body fluids and cell culture media.

A sample, consisting of whole blood (in EDTA, Heparin or ACD) ordissociated tissue or body fluids (synovial fluid) or cell culture mediais obtained and stored in a manner so as not to decrease viability. Thesample is washed sufficiently to remove plasma, media, body fluid,debris and extra-cellular enzymes. The wash media consists of aphysiologically balanced buffered salt solution. The washed cells areincubated at 37° C. 50 μL of sample and 25 μL of substrate media aremixed together and allowed to incubate at 37° C. for a predeterminedamount of time. At the end of the incubation period, the sample is thenmeasured for fluorescence.

Process D: Examination of platelets, erythrocytes, leukocytes,dissociated tissue, body fluids and cell culture media using amechanical separation to isolate a cell population.

A sample, consisting of whole blood (in EDTA, Heparin or ACD) ordissociated tissue or body fluids (synovial fluid) or cell culture mediais obtained and stored in a manner so as not to decrease viability. Amechanical separation to isolate a specific cell population isperformed, i.e., ficoll, differential centrifugation, differentialprecipitation. The sample is washed sufficiently to remove plasma,media, body fluid, debris and extra-cellular enzymes. The wash mediaconsists of a physiologically balanced buffered salt solution. Thewashed cells are incubated at 37° C. 50 μL of sample and 25 μL ofsubstrate media are mixed together and allowed to incubate at 37° C. fora predetermined amount of time. At the end of the incubation period, thesample is then measured for fluorescence.

2. Detection of Fluorescence

The instruments used to detect fluorescence are the flow cytometer orfluorescent microscope. There are four different instrumentconfigurations for the flow cytometer, A, B, C and D. Any of the fourconfigurations can be used with any one of the sample preparationsdescribed above. The choice of which configuration is selected isdependent upon the user and the information sought to be obtained. Thefour configurations are:

Configuration A

Configuration A analyzes the cells by size, granularity and singlecolor. In the first configuration, the flow cytometer separates thecells by size and granularity. The activity of an enzyme is then assayedusing the reagent compound. Two samples are allowed to proceed atdifferent times and the reaction is stopped. The difference influorescence permits the calculation of a rate. Total population countspreferred are 20,000 to 500,000 cells. Use of light scatter orhematology parameters provide size and granularity separation. Intensitybitmap of desired populations and determination of fluorescent activityby single measurement point or multi-point measurement can be employed.Determine count, percentage and fluorescent intensity of a multi-modalpopulation representing enzymatic activity.

Configuration B

Configuration B analyzes the cells by size, granularity and two colors.In the second configuration, the flow cytometer separates the cells bysize and granularity. Cell morphology is determined by a fluorescenceassay with a monoclonal antibody marker. The rate of the hydrolysis ofthe assay compound is then determined. Total population counts preferredare 20,000 to 500,000 cells. Use of light scatter or hematologyparameters provide size and granularity separation. Intensity bitmap ofdesired populations and determination of fluorescent activity by singlemeasurement or multi-point measurement can be employed. Determine count,percentage and fluorescent intensity of a multi-modal populationrepresenting enzymatic activity. The analysis is a 2-color analysismeasuring enzymatic activity in one color and surface-marker antibodycell morphology in the other color.

Configuration C

Configuration C analyzes the cells by size, granularity, two colors andbackgate fluorescence. Configuration 3 is a modification of the Duquemethod. Duque, R. E., "Flow Cytometric Analysis of Lymphomas and AcuteLeukemias", Annals of the New York Academy of Sciences, Clinical FlowCytometry, 677, pp. 309-325 (Mar. 20, 1993). The size and granularity ofthe cell are separated by a flow cytometer using light scatter and/orwith surface markers, such as monoclonal antibodies. A series of cellpopulations are determined, with rearrangement of the histogram toidentify the disease and normal cells. The activity of the enzyme isthen assayed. Total population counts preferred are 20,000 to 500,000cells. Use of light scatter or hematology parameters provide size andgranularity separation. Intensity bitmap of desired populations anddetermination of fluorescent activity by single measurement point ormulti-point measurement can be employed. Determine count, percentage andfluorescent intensity of a multi-modal population representing enzymaticactivity. The analysis is a 2-color analysis measuring enzymaticactivity in one color and surface-marker antibody cell morphology in theother color. Backgate fluorescence data on size and granularity todetermine count and percent of diseased cells.

Configuration D

Configuration D analyzes activity of a population of cells over time.Total population counts preferred are 20,000 to 500,000. Use of lightscatter or hematology parameters provide Size and granularityseparation. Intensity bitmap of desired populations and determination offluorescent activity by single measurement point or multi-pointmeasurement can be employed. Determine count, percentage and fluorescentintensity of a multi-modal population representing enzymatic activity.The analysis is a 2-color analysis measuring enzymatic activity in onecolor and surface-marker antibody cell morphology in the other color.

3. Data Analysis

The measured fluorescence intensity can be converted from mean channelfluorescence (in peak or integrated mode) to MESF (molecules ofequivalent soluble fluorochrome, Flow Cytometry Standards Corp., SanJuan, Puerto Rico) or International Units of hydrolysis per cell. Anormal range of enzyme activity is established by assaying males andfemales in sufficient quantity to characterize the population levelsstatistically.

Various disease states are assayed for enzymatic activity and comparedto the normal range. Three conditions will exist from this data:

1. Obvious increases or decreases in enzyme levels outside the normalrange

2. Patterns of enzyme activities representing morphology

3. Patterns of enzyme activities representing disease states

Artificial intelligence or Non-Negative Least Squares (NNLS) programsand analysis of variance (ANOVA) programs are useful in identifyingpatterns of enzyme activities representing morphology, cell types andpatterns of enzyme activities representing disease states.

A first, and most obvious technique for disease diagnosis is identifyingthe absence or presence of a single enzyme. One example of such a singleenzyme diagnosis is the diagnosis for Gaucher's disease, which isdiagnosed depending on the lack of a particular enzyme, namely,glucocerebrosidase.

The remaining techniques treat the absence or presence of a combinationof enzymes as a complex interplay of metabolic systems, wherein eachcell contains a group of enzymes, and the concentrations of these groupsof enzymes are gaussian distributed, having a normal range or values,wherein values in disease states fall outside the normal range.

Analysis of Variance (ANOVA)

There are two steps in the mathematical analysis of the data. The firststep is to analyze the variance in the data. The purpose of the analysisis to identify which combination of enzyme concentrations for variouscell types are diagnostic of particular disease states or treatmentmodalities. (For simplicity, disease states and treatment modalitieswill be referred to collectively as disease states, henceforth).

To analyze the variance, each set of enzyme concentrations for eachenzyme measured for each cell type is considered as a component of acomposite measurement vector. A data matrix, such as the Full CovarianceData Matrix in FIG. 14A, including columns of basis measurement vectorsknown to characterize certain disease states is generated. The rows ofthe matrix represent the measurement vector components for each diseasestate under consideration. A variation across a row indicates that thevarious disease states affect cell metabolism such that theconcentration of that enzyme in that cell type is changed; obviouslysuch a difference provides information about the underlying diseasestate.

The data matrix needs to be developed for different disease states.Patients for the disease state data matrix can be first identified usingconventional technologies. The disease state matrix can be expanded toinclude differentiation of stages of the disease as well as theinfluence of drug pharmokinetics on cellular function. Separate studiesof drug pharmokinetics on human tissue culture cell lines can beperformed to provide a reference.

The metabolism controlled by some enzymes in some cell types will beinsensitive to disease state. Others will react collectively in acomplex pattern with different enzymes in certain cells to produce apattern of enzyme concentrations that will definitively characterize aparticular disease state. The analysis of variance is necessary toselect out that combination of enzymes in particular cells which aremost useful in distinguishing among the various disease states spannedby the basis measurement vectors.

The selection of a relatively small number of components for themeasurement vector is necessary to simplify the later analysis, reducethe number of physical measurements which must be made and to reduce theeffects of spurious noise generated from the measurements and fromindividual variation among the same population. An example of theselected number of components is illustrated in FIG. 14A as the ReducedCovariance Data Matrix of Strongly Contributing Factors.

Squared Deviations From the Mean

The simplest way to analyze the variance is to compute the varianceacross the row for each row in the data matrix. Those rows with a highvariance correspond to enzymes whose concentration in a particular celltype varies most strongly across the disease states under consideration.This method of analysis neglects any interaction of various enzymes witheach other; however, it provides a simple, gross indication of whichenzymes in which cell types are most affected by the disease state. Thistechnique was used in the "Variance" column of Table 5.

Eigenvector or Principal Components Analysis of Variance

A more complete way to analyze variance is to compute the eigenvaluesand eigenvectors of the data matrix, as described for example in J. D.Jobson, Applied Multivariate Data Analysis, Springer Verlag N.Y. (1992).Such an analysis is conventionally termed an eigenvector analysis or aprincipal components analysis (PCA). In practice, rather than on thedata matrix, D, itself, the eigenvector analysis is performed on thecovariance matrix of the data (D-m)^(t) (D-m), where the superscript trefers to the transpose of the matrix and m is the vector whosecomponents are the mean of each row of D. Each resulting eigenvectorincludes a particular weighted combination of the measurement vectorcomponents which act in concert with each other. Each eigenvector has acorresponding eigenvalue which is proportional to the total variance inthe measurement which is accounted for by that eigenvector. Eacheigenvector is also orthogonal or independent of every othereigenvector. This technique was used in the "Eigenvector 1" and"Eigenvector 2" columns of Table 5.

For the data matrices here, if the eigenvectors are arranged in order ofdecreasing eigenvalue, the vast majority of the variance is accountedfor by the first several eigenvectors. Thus, the principal factorsdistinguishing the various disease states under consideration can becaptured with a small number of combinations of the enzymeconcentrations in the various cell types. FIG. 14A illustrates theprocess of developing the reduced covariance data matrix by eigenvectoranalysis.

An examination of the combination of measurement vector componentscontributing most to distinguishing different disease states, in lightof the metabolic pathways linking the various enzymes, can be used tounderstand the underlying metabolic changes occurring with the variousdisease states. The foregoing eigenvector analysis is used to selectfrom the entire series of measured enzyme concentrations for differentcell types, the combination of enzymes which is most useful incharacterizing the disease state.

Diagnosing Disease State, Based on Measurement NNLS

Once the measurement vector and data matrix are reformed with theselected (reduced number of) enzyme concentrations for particular celltypes, measurements of patients with unknown diseases (presumed to bewithin the basis disease states of the data matrix) can be used todiagnose their disease state. Two methods are described here toaccomplish the second step of the mathematical analysis of the data, theinference of disease state from the measurement vector: a Non-NegativeLeast Squares based algorithm (NNLS) and a neural net.

NNLS is a non-negatively constrained least squares solution to theproblem of determining the disease state from the reduced measurementvector. The algorithm, which is disclosed in C. L. Lawson and R. J.Hanson, Solving Least Squares Problems, Prentice-Hall N.J. (1974) findsthat linear combination of the basis measurement vectors which mostclosely fits, in a least squares sense, the measurement vector of thepatient whose state is being diagnosed. The resulting solution is avector the magnitude of whose components reflect the probability thatthe unknown disease state is each one of the basis disease states. Thealgorithm constrains the components of the solution vector to benon-negative; this constraint can be applied because the components ofthe vector represent probabilities, which by definition must benon-negative. The non-negativity constraint is extremely important instabilizing the solution to this often mathematically ill-conditionedinversion problem.

Ideally, the solution vector has only a single non-zero component, inthis case, the disease state corresponding to the chosen basismeasurement vector is the diagnosis of the patient. Because of noise inthe measurements and individual biological variation among individualsor bemuse a patient is afflicted with more than one disease at a time,the solution vector provides a range of possibilities for the diagnosis;the component with the highest magnitude representing the most probabledisease state, etc. FIG. 14B illustrates the process of extractingpredicted disease state probabilities from the reduced covariance datamatrix.

As illustrated in FIG. 14A, a database is obtained for both normalstates and various disease states. An example of an array of valuesobtained for 5 cell types (lymphocytes, monocytes, platelets,granulocytes and erythrocytes) using 50 different enzyme assays is shownin Table 3A-3C. This Table further gives a mean value, two standarddeviation low value and two standard deviation high value for each cellfor each enzyme.

                                      TABLE 3A                                    __________________________________________________________________________    NORMAL LEUKOCYTE ENZYME ACTIVITY                                                                  LYMPHS      MONOS       GRANS                                                 Mean                                                                              Lo  Hi  Mean                                                                              Lo  Hi  Mean                                                                              Lo  Hi                        __________________________________________________________________________    Aminopep   LEU      34.94                                                                             14.73                                                                             55.14                                                                             120.66                                                                            35.52                                                                             205.80                                                                            80.19                                                                             28.42                                                                             131.96                    Aminopep M ALA      75.85                                                                             47.46                                                                             104.25                                                                            172.33                                                                            107.51                                                                            237.15                                                                            149.02                                                                            85.05                                                                             212.98                    Pro Aminopep                                                                             PRO      1.70                                                                              0.53                                                                              2.88                                                                              6.83                                                                              2.62                                                                              11.04                                                                             9.19                                                                              3.96                                                                              14.41                     Aminopep M LYS      1.16                                                                              0.33                                                                              1.99                                                                              7.56                                                                              3.83                                                                              11.29                                                                             4.27                                                                              0.00                                                                              8.88                      Aminopep M, N                                                                            GLY      18.18                                                                             7.21                                                                              29.14                                                                             81.47                                                                             30.13                                                                             92.82                                                                             29.48                                                                             9.82                                                                              49.14                     Aminopep N SER      1.35                                                                              0.28                                                                              2.41                                                                              6.49                                                                              2.71                                                                              10.26                                                                             3.40                                                                              0.75                                                                              6.06                      Endopeptidase I                                                                          Z-ARG    2.02                                                                              0.67                                                                              3.38                                                                              16.80                                                                             7.83                                                                              25.78                                                                             7.22                                                                              0.37                                                                              14.06                     Endopeptidase I                                                                          ARG      1.47                                                                              0.50                                                                              2.45                                                                              10.34                                                                             5.13                                                                              15.54                                                                             5.16                                                                              0.23                                                                              10.10                     Aminopep A ASP      0.26                                                                              0.04                                                                              0.49                                                                              0.66                                                                              0.06                                                                              1.27                                                                              0.45                                                                              0.01                                                                              0.90                      Cathepsin B                                                                              VS       3.81                                                                              0.57                                                                              7.06                                                                              19.61                                                                             2.86                                                                              36.36                                                                             8.27                                                                              2.68                                                                              13.87                     Cathepsin B                                                                              VS-M     3.78                                                                              0.86                                                                              6.70                                                                              31.14                                                                             5.17                                                                              57.11                                                                             7.07                                                                              1.75                                                                              12.40                     Cathepsin B                                                                              VK       1.87                                                                              0.05                                                                              3.69                                                                              20.38                                                                             0.00                                                                              54.92                                                                             5.10                                                                              0.65                                                                              9.54                      Cathepsin B                                                                              VK-M     2.05                                                                              0.00                                                                              4.40                                                                              43.37                                                                             6.71                                                                              80.04                                                                             4.31                                                                              0.00                                                                              8.76                      Cathepsin B                                                                              QS       1.70                                                                              1.06                                                                              2.34                                                                              10.66                                                                             4.02                                                                              17.30                                                                             4.67                                                                              2.08                                                                              7.25                      Cathepsin B                                                                              QS-M     1.14                                                                              0.67                                                                              1.61                                                                              8.69                                                                              2.81                                                                              14.57                                                                             2.71                                                                              1.43                                                                              4.00                      Cathepsin B                                                                              LG       13.83                                                                             6.56                                                                              21.09                                                                             44.56                                                                             22.50                                                                             66.62                                                                             29.99                                                                             11.28                                                                             48.69                     Cathepsin B                                                                              LG-M     4.71                                                                              1.33                                                                              8.08                                                                              23.92                                                                             0.00                                                                              48.54                                                                             10.54                                                                             3.83                                                                              17.26                     Dipep Peptidase II                                                                       KA       11.47                                                                             3.00                                                                              19.93                                                                             39.35                                                                             16.82                                                                             61.88                                                                             29.15                                                                             9.25                                                                              49.05                     Dipep Peptidase II                                                                       KA-M     0.55                                                                              0.30                                                                              0.80                                                                              9.74                                                                              1.47                                                                              16.01                                                                             1.74                                                                              0.82                                                                              2.65                      Dipep Peptidase IV                                                                       Z-AA     151.20                                                                            113.68                                                                            188.73                                                                            328.25                                                                            243.23                                                                            413.27                                                                            363.20                                                                            273.63                                                                            452.87                    Dipep Peptidase IV                                                                       Z-AA-M   94.47                                                                             47.80                                                                             141.14                                                                            210.71                                                                            112.01                                                                            309.42                                                                            252.24                                                                            138.76                                                                            365.72                    Dipep Peptidase IV                                                                       Z-GP     107.56                                                                            72.84                                                                             142.28                                                                            225.10                                                                            155.83                                                                            294.37                                                                            240.99                                                                            148.98                                                                            332.99                    Dipep Peptidase IV                                                                       Z-GP-M   53.74                                                                             29.05                                                                             78.44                                                                             119.81                                                                            70.14                                                                             169.48                                                                            130.62                                                                            63.36                                                                             197.87                    Cathepsin D                                                                              GL       9.42                                                                              3.84                                                                              15.00                                                                             41.78                                                                             20.02                                                                             63.54                                                                             29.16                                                                             11.99                                                                             46.33                     Cathepsin D                                                                              GL-M     2.56                                                                              0.00                                                                              5.34                                                                              89.11                                                                             0.00                                                                              195.37                                                                            12.01                                                                             0.00                                                                              27.36                     Cathepsin C                                                                              Z-AG     15.17                                                                             7.48                                                                              22.86                                                                             34.51                                                                             18.95                                                                             50.08                                                                             33.19                                                                             17.20                                                                             49.18                     Cathepsin C                                                                              Z-AG-M   14.24                                                                             2.97                                                                              25.52                                                                             37.75                                                                             12.88                                                                             62.61                                                                             34.70                                                                             9.31                                                                              60.09                     Dipep Peptidase IV                                                                       AA-TFA   56.95                                                                             3.01                                                                              110.89                                                                            158.31                                                                            31.98                                                                             284.63                                                                            154.81                                                                            25.75                                                                             283.87                    Dipep Peptidase IV                                                                       AA-M     9.88                                                                              0.00                                                                              24.56                                                                             58.29                                                                             1.79                                                                              114.79                                                                            29.91                                                                             9.29                                                                              50.53                     Cathepsin D                                                                              Z-TP 6.5 21.97                                                                             8.98                                                                              34.96                                                                             49.69                                                                             22.25                                                                             77.13                                                                             58.05                                                                             24.15                                                                             91.96                     Cthepsin D Z-TP 6.5-M                                                                             20.62                                                                             9.03                                                                              32.22                                                                             45.70                                                                             21.61                                                                             69.90                                                                             55.07                                                                             23.22                                                                             86.91                     Cathepsin B                                                                              LLR      4.09                                                                              0.00                                                                              9.90                                                                              54.32                                                                             28.31                                                                             80.32                                                                             9.46                                                                              0.00                                                                              23.73                     Cathepsin B                                                                              LLR-M    3.38                                                                              0.50                                                                              6.26                                                                              62.85                                                                             14.37                                                                             111.33                                                                            7.58                                                                              0.00                                                                              24.34                     Cathepsin B                                                                              LGLG     1.42                                                                              0.76                                                                              2.08                                                                              11.36                                                                             0.00                                                                              23.64                                                                             4.08                                                                              1.93                                                                              6.23                      Cathepsin B                                                                              LGLG-M   1.34                                                                              0.38                                                                              2.29                                                                              40.74                                                                             0.00                                                                              98.16                                                                             3.24                                                                              1.12                                                                              5.37                      Estertase  FDA      13.38                                                                             0.00                                                                              27.21                                                                             66.25                                                                             17.06                                                                             115.43                                                                            68.37                                                                             12.90                                                                             123.84                    Monocytic Esterase                                                                       FDA-NAF  17.98                                                                             0.43                                                                              35.54                                                                             93.36                                                                             0.00                                                                              210.77                                                                            81.99                                                                             17.39                                                                             146.60                    Peroxidase DCFH-DA MES                                                                            4.11                                                                              0.68                                                                              7.53                                                                              12.28                                                                             2.23                                                                              22.34                                                                             7.49                                                                              0.95                                                                              14.04                     Activated Peroxidase                                                                     DCFH MES PMA                                                                           4.69                                                                              0.98                                                                              8.41                                                                              22.18                                                                             0.00                                                                              45.35                                                                             11.02                                                                             0.00                                                                              28.46                     Collagenase                                                                              GPLGP    8.97                                                                              3.64                                                                              14.30                                                                             23.38                                                                             10.98                                                                             35.79                                                                             16.88                                                                             6.80                                                                              26.96                     Collagenase                                                                              GPLGP-M  7.89                                                                              2.40                                                                              13.37                                                                             23.32                                                                             10.36                                                                             36.28                                                                             13.82                                                                             4.39                                                                              23.24                     Collagenase                                                                              GPGA     0.36                                                                              0.22                                                                              0.60                                                                              1.01                                                                              0.01                                                                              2.01                                                                              1.00                                                                              0.36                                                                              1.63                      Elastase   RGES     1.43                                                                              0.75                                                                              2.12                                                                              8.37                                                                              4.28                                                                              12.46                                                                             3.96                                                                              2.09                                                                              5.83                      Glucosidase                                                                              DIGLUC   0.94                                                                              0.42                                                                              1.46                                                                              6.58                                                                              0.25                                                                              10.91                                                                             2.35                                                                              0.10                                                                              4.60                      Acid Phosphatase                                                                         PO4 5.0  54.29                                                                             17.08                                                                             91.49                                                                             148.32                                                                            61.42                                                                             235.23                                                                            85.42                                                                             13.42                                                                             157.42                    Galactidase                                                                              DIGALAC  0.38                                                                              0.00                                                                              1.05                                                                              5.94                                                                              0.00                                                                              17.90                                                                             1.58                                                                              0.00                                                                              4.32                      Cathepsin C                                                                              Z-TP 8.7-M                                                                             30.99                                                                             16.24                                                                             45.75                                                                             71.45                                                                             38.94                                                                             103.97                                                                            80.16                                                                             45.97                                                                             114.35                    Cathepsin C                                                                              Z-TP 8.7 35.01                                                                             24.75                                                                             45.26                                                                             76.00                                                                             53.89                                                                             98.11                                                                             82.35                                                                             59.36                                                                             105.33                    Glucosidase                                                                              DIGLUC (FL)                                                                            0.23                                                                              0.15                                                                              0.32                                                                              1.89                                                                              0.00                                                                              4.55                                                                              0.45                                                                              0.07                                                                              0.83                      Neutral Butyrate                                                                         DIBUT 7.5                                                          Acidic Butyrate                                                                          DIBUT 6.5                                                                              4.08                                                                              0.00                                                                              12.16                                                                             10.37                                                                             0.00                                                                              30.20                                                                             6.43                                                                              0.00                                                                              18.24                     Esterase   CHLOAC   2.49                                                                              0.85                                                                              4.13                                                                              7.33                                                                              1.76                                                                              12.89                                                                             6.48                                                                              1.40                                                                              11.57                     Esterase   DIACET 6.5                                                                             109.89                                                                            39.37                                                                             180.41                                                                            534.00                                                                            322.46                                                                            745.54                                                                            408.88                                                                            251.29                                                                            566.46                    Esterase   DIACET 7.5                                                                             89.30                                                                             0.00                                                                              219.98                                                                            432.78                                                                            0.00                                                                              986.33                                                                            348.01                                                                            0.00                                                                              781.34                    Acidic Prop Esterase                                                                     DIPRO 6.5                                                                              86.11                                                                             0.00                                                                              216.26                                                                            441.65                                                                            0.00                                                                              1040.17                                                                           349.18                                                                            0.00                                                                              838.78                    Neutral Prop Esterase                                                                    DIPRO 7.6                                                                              95.34                                                                             0.00                                                                              219.08                                                                            489.27                                                                            0.00                                                                              1106.82                                                                           429.17                                                                            0.00                                                                              977.31                    Acidic Valerate Esterase                                                                 DIVAL 6.5                                                                              33.13                                                                             20.42                                                                             45.84                                                                             184.51                                                                            119.00                                                                            250.02                                                                            92.81                                                                             47.67                                                                             137.95                    Acidic Hex Esterase                                                                      DIHEX 6.5                                                                              15.10                                                                             4.22                                                                              25.97                                                                             72.87                                                                             24.73                                                                             121.00                                                                            33.84                                                                             11.39                                                                             56.29                     Neutral Hex Esterase                                                                     DIHEX 7.5                                                                              7.54                                                                              0.00                                                                              15.15                                                                             37.22                                                                             10.06                                                                             64.37                                                                             17.57                                                                             2.89                                                                              32.24                     Acidic Hep Esterase                                                                      DIHEP 6.5                                                                              9.17                                                                              0.00                                                                              19.35                                                                             32.25                                                                             3.51                                                                              60.99                                                                             18.57                                                                             3.86                                                                              33.28                     Neutral Hep Esterase                                                                     DIHEP 7.5                                                                              9.73                                                                              0.00                                                                              19.52                                                                             36.15                                                                             7.40                                                                              64.89                                                                             20.41                                                                             0.92                                                                              39.89                     Acidic Pal Esterase                                                                      DIPAL 6.5                                                                              0.15                                                                              0.00                                                                              0.31                                                                              0.30                                                                              0.16                                                                              0.45                                                                              0.14                                                                              0.09                                                                              0.20                      Neutral Pal Esterase                                                                     DIPAL 7.5                                                                              0.14                                                                              0.00                                                                              0.29                                                                              0.30                                                                              0.13                                                                              0.47                                                                              0.14                                                                              0.10                                                                              0.19                      __________________________________________________________________________

                  TABLE 3B                                                        ______________________________________                                        NORMAL ERYTHROCYTE ENZYME ACTIVITY                                            SUBSTRATE ENZYME         MEAN    2SD LO                                                                              2SD HI                                 ______________________________________                                        LEU       Aminopeptidase 0.107   0.105 0.108                                  ALA       Aminopeptidase M                                                                             0.237   0.000 0.602                                  PRO       Pro Aminopeptidase                                                                           0.121   0.087 0.155                                  LYS       Aminopeptidase M                                                                             0.109   0.099 0.118                                  GLY       Aminopeptidase M, N                                                                          0.163   0.025 0.300                                  SER       Aminopeptidase N                                                                             0.107   0.107 0.107                                  Z-ARG     Enodpeptidase I                                                                              0.106   0.106 0.106                                  ARG       Endopeptidase I                                                                              0.106   0.104 0.107                                  ASP       Aminopeptidase A                                                                             0.106   0.106 0.106                                  VS        Cathepsin B    0.138   0.073 0.203                                  VS-M      Cathepsin B    0.176   0.027 0.324                                  VK        Cathepsin B    0.170   0.097 0.242                                  VK-M      Cathepsin B    0.129   0.093 0.164                                  QS        Cathepsin B    0.121   0.108 0.133                                  QS-M      Cathepsin B    0.116   0.113 0.119                                  LG        Cathepsin B    0.138   0.080 0.195                                  LG-M      Cathepsin B    0.131   0.129 0.132                                  KA        Dipeptidylpeptidase II                                                                       0.118   0.096 0.139                                  KA-M      Dipeptidylpeptidase II                                                                       0.110   0.104 0.116                                  Z-AA      Dipeptidylpeptidase IV                                                                       0.484   0.000 1.454                                  Z-AA-M    Dipeptidylpeptidase IV                                                                       0.411   0.000 1.227                                  Z-GP      Dipeptidylpeptidase IV                                                                       0.438   0.000 0.976                                  Z-GP-M    Dipeptidylpeptidase IV                                                                       0.334   0.000 0.818                                  GL        Cathepsin D    0.190   0.111 0.269                                  GL-M      Cathepsin D    0.286   0.000 0.734                                  Z-AG      Cathepsin C    0.225   0.000 0.482                                  Z-AG-M    Cathepsin C    0.171   0.115 0.226                                  AA-TFA    Dipeptidylpeptidase IV                                                                       0.144   0.070 0.218                                  AA-M      Dipeptidylpeptidase IV                                                                       0.131   0.097 0.165                                  Z-TP 6.5  Cathepsin C    0.132   0.098 0.166                                  Z-TP 6.5M Cathepsin C    0.168   0.070 0.265                                  LLR       Cathepsin B    0.225   0.000 0.529                                  LLR-M     Cathepsin B    0.476   0.000 1.492                                  LGLG      Cathepsin B    0.122   0.091 0.153                                  LGLG-M    Cathepsin B    0.150   0.059 0.241                                  FDA       Esterase       0.126   0.089 0.163                                  FDA-NAF   Monocytic Esterase                                                                           0.136   0.088 0.184                                  DCFH-DAMES                                                                              Peroxidase     0.286   0.000 0.764                                  DCHFMESPMA                                                                              Activated peroxidase                                                                         0.268   0.000 0.693                                  GPLGP     Collagenase    0.164   0.136 0.192                                  GPLGP-M   Collagenase    0.119   0.103 0.134                                  GFGA      Collagenase    0.131   0.073 0.188                                  RGES      Elastase       0.118   0.098 0.138                                  DGLUC 49  Glucosidase    0.114   0.109 0.118                                  DPO4 46   Acid Phosphatase                                                                             0.151   0.090 0.211                                  GALAC 50  Galactidase    0.119   0.111 0.127                                  TP 8.7M 48                                                                              Cathepsin C    0.212   0.000 0.467                                  TP 8.7 47 Cathepsin C    0.161   0.054 0.267                                  FL GLUC   Glucosidase    0.110   0.110 0.110                                  ______________________________________                                    

                  TABLE 3C                                                        ______________________________________                                        NORMAL PLATELET ENZYME ACTIVITY                                               SUBSTRATE ENZYME         MEAN    2SD LO                                                                              2SD HI                                 ______________________________________                                        LEU       Aminopeptidase 1.006   0.640 2.156                                  ALA       Aminopeptidase M                                                                             1.726   0.846 3.422                                  PRO       Pro Aminopeptidase                                                                           0.295   0.111 0.854                                  LYS       Aminopeptidase M                                                                             0.232   0.110 0.440                                  GLY       Aminopeptidase M, N                                                                          3.622   0.897 8.030                                  SER       Aminopeptidase N                                                                             0.309   0.135 0.579                                  Z-ARG     Enodpeptidase I                                                                              0.262   0.133 0.448                                  ARG       Endopeptidase I                                                                              0.268   0.128 0.499                                  ASP       Aminopeptidase A                                                                             0.403   0.170 1.274                                  VS        Cathepsin B    0.576   0.175 2.007                                  VS-M      Cathepsin B    0.592   0.180 1.622                                  VK        Cathepsin B    0.839   0.144 3.080                                  VK-M      Cathepsin B    0.486   0.153 1.343                                  QS        Cathepsin B    0.481   0.186 1.176                                  QS-M      Cathepsin B    0.499   0.280 1.194                                  LG        Cathepsin B    0.470   0.182 0.922                                  LG-M      Cathepsin B    0.438   0.143 1.032                                  KA        Dipeptidylpeptidase II                                                                       0.447   0.161 1.034                                  KA-M      Dipeptidylpeptidase II                                                                       0.277   0.126 0.562                                  Z-AA      Dipeptidylpeptidase IV                                                                       1.372   0.480 3.438                                  Z-AA-M    Dipeptidylpeptidase IV                                                                       1.199   0.545 3.253                                  Z-GP      Dipeptidylpeptidase IV                                                                       2.668   0.864 5.867                                  Z-GP-M    Dipeptidylpeptidase IV                                                                       1.415   0.481 3.883                                  GL        Cathepsin D    0.702   2.010 1.930                                  GL-M      Cathepsin D    0.511   0.254 1.235                                  Z-AG      Cathepsin C    7.900   0.411 21.322                                 Z-AG-M    Cathepsin C    5.113   0.280 13.295                                 AA-TFA    Dipeptidylpeptidase IV                                                                       0.622   0.218 1.274                                  AA-M      Dipeptidylpeptidase IV                                                                       0.350   0.145 0.684                                  Z-TP 6.5  Cathepsin C    2.105   0.251 4.841                                  Z-TP 6.5M Cathepsin C    2.348   0.347 5.889                                  LLR       Cathepsin B    2.150   0.484 5.562                                  LLR-M     Cathepsin B    0.434   0.239 0.886                                  LGLG      Cathepsin B    2.275   0.230 10.086                                 LGLG-M    Cathepsin B    4.013   0.211 9.648                                  FDA       Esterase       15.353  2.960 29.955                                 FDA-NAF   Monocytic Esterase                                                                           7.201   2.310 14.369                                 DCFH-DAMES                                                                              Peroxidase     0.764   0.317 1.840                                  DCFH-DAATRIS                                                                            Super oxide dismutase                                                                        3.556   1.640 7.982                                  DCHFMESPMA                                                                              Activated peroxidase                                                                         1.979   0.576 7.045                                  DCFHTRISPMA                                                                             Activated super oxide                                                                        5.111   1.870 12.045                                           dismutase                                                           ______________________________________                                    

Artificial Intelligence By Back Propagation (Neural Net)

An alternate method of analyzing the data is via neural net.

To determine the interrelationship of enzyme function in both the celland cell type, the ratios of enzyme activities needs to be analyzed. Tofully analyze all possible combinations of a data set, an artificialintelligence system such as "Neuroshell™" (Ward Systems Group Inc.,Frederick, Md.) may be used.

The basic building block of artificial intelligence neural networktechnology is the simulated neuron, which processes a number of inputsto produce an output. Inputs and outputs are numeric values between 0and 1 which represents positive stimulation close to 1 and negativestimulation close to 0. Inputs are data entered and outputs either comefrom other neurons or are displayed as results. The process by which theneuron processes its inputs to arrive at an output is usually asummation of inputs followed by a linear function applied to the sum.Independent neurons are of little use unless connected to a network ofneurons called nodes. Nodes are layered and interconnected to receiveinformation from each other. As each input node passes information toeach other and the next layer, the values are weighed to represent theconnection strength. To positively reinforce a connection the weight israised and likewise to negatively reinforce or inhibit a connection theweight is lowered. The network processes data by accepting inputpatterns into input nodes or Defining Characteristics. The networkproduces output patterns which are called Classifying Characteristics.The user of the algorithm can adjust the output pattern by adjustingoutput thresholds. Feedback from the user determines whether thereinforcement is positive or negative. Learning in a neural networkoccurs when a set of input patterns (cell type and enzyme concentration)is given with a known output pattern (Disease state or Normal). This iscalled a sample case. The error between the predicted and actual outputsfor a given output node is measured and the total error is one-half ofthe sum of the squares of the difference. The weights leading to thisoutput node are modified slightly (specified by the user as learningrate, a percentage of the error to be used in the next iteration) duringeach iteration of a learning session in the direction required toproduce a smaller error the next time the same pattern is presented.This is how the neural network "learns". Learning is continued until theconsummate error of all output nodes falls below a learning thresholdcontrolled by the user. Upon completion of learning, the network shouldbe capable of reproducing the correct output pattern (disease or normal)when presented with one of the input patterns it has learned. Moreover,the network is capable of generalizing by recognizing an input patternclose to a pattern it has learned and produces an output close to apattern it was trained to produce. A simple two layered network isincapable of learning complex patterns. Back propagation uses one ormore layers of hidden nodes and a nonlinear function algorithm Theweight applied to the nodes must then be back propagated through alllayers of the nodes. The number of hidden nodes is determined by theuser for a specific problem. If too few hidden nodes are used, then allthe unique situations found in the sample case will not be explored andif too many are used, learning will never complete. The neural networkapproach provides the opportunity to look at ratios of cell types andenzyme levels in all possible combinations for both disease and healthusing a simple format.

Progression of a disease during treatment to monitor "return tonormalcy" or further increase in stage or complication with additionaldisease states can be done by monitoring the NNLS predictive diseaseprobabilities over time or the value of the Neural Network score as itapproaches normalcy or the three-dimensional plotting of cell-typeenzyme activity patterns comparison to normal. Recurrent Neural Networksmay also be used for time series data. Examples of these types are theProbabilistic Neural Network (PNN), General Regression Neural Network(GRNN) and the Kohonen-Realty Neural Network.

One embodiment of an analysis system using neural networks isillustrated in FIG. 15. The same database illustrated in FIG. 14A usingthe ANOVA technique is also used in the neural network implementation.The database is input into this neural network has a training set andthe user sets a threshold, learning rate, and learning momentum. Oncethe neural network has "learned" on the plurality of sample cases, atest case is input to the neural network including an unknown to beclassified as a disease state or normal state. Based on what the neuralnetwork as learned with a plurality of sample cases, the neural networkoutputs the best predictor of the test case. The neural network may alsobe utilized to perform time course monitoring of an individual patientfor return to normalcy, using an advanced probabilistic neural network(TNN) program.

Look-Up Tables

An alternative to the NNLS and Neural Net analyses described above usesLook-Up tables and is similar to the Expert System described in "WhiteCell and Thrombocyte Disorders--Standardized Self-learning FlowCytometric List Mode Data Classification with the CLASSIF1 ProgramSystem", Valet et al, Ann. N.Y. Acad. Sci., 677: 233-251 (1993).

Definitions

As used herein, either individually or as part of a larger group,"alkyl" means a linear, cyclic, or branched-chain aliphatic moiety ofone to 10 carbon atoms; "substituted alkyl" means an alkyl group havinga substituent containing a heteroatom or heteroatoms such as N, O, or S;"aryl" means an aromatic moiety, e.g., phenyl, of 6 to 18 carbon atoms,unsubstituted or substituted with one or more alkyl, substituted alkyl,nitro, alkoxy, or halo groups; and "alkaryl" means an aryl moiety of 7to 19 carbons having an aliphatic substituent, and optionally, othersubstituents such as one or more alkyl, substituted alkyl, alkoxy oramino groups. "Aralkyl" means a linear or branched-chain aliphaticmoiety of six to 18 carbon atoms comprising an aryl group or groups.

The following common chemical abbreviations are used in the examples:

t-BOC=tertiarybutyloxycarbonyl

EDAC=1-ethyl-3-(3'-dimethylaminopropylcarbodiimide)-hydrochloride

FMOC=9-fluorenylmethyloxycarbonyl

BOP=benzotriazoly-N-oxy-tris(dimethylamino)-phosphonium-hexafluorophosphate

HBOT=1-hydroxybenzotriazole

HPLC=High pressure liquid chromatography

TLC=Thin layer chromatography

V:V=Volume to volume

The amino acids are abbreviated as follows:

    ______________________________________                                        Amino Acid           Abbreviation                                             ______________________________________                                        L-alanine            Ala or A                                                 L-arginine           Arg or R                                                 L-asparagine         Asn or N                                                 L-aspartic acid      Asp or D                                                 L-cysteine           Cys or C                                                 L-glutamic acid      Glu or E                                                 L-glutamine          Gln or Q                                                 glycine              Gly or G                                                 L-histidine          His or H                                                 L-isoleucine         Ile or I                                                 L-leucine            Leu or L                                                 L-lysine             Lys or K                                                 L-methionine         Met or M                                                 L-phenylalanine      Phe or F                                                 L-proline            Pro or P                                                 L-serine             Ser or S                                                 L-threonine          Thr or T                                                 L-tryptophan         Trp or W                                                 L-tyrosine           Tyr or X                                                 L-valine             Val or V                                                 ______________________________________                                    

The synthesis of the assay compounds can be further understood byreference to the Examples. It will be appreciated, however, that theinvention is not limited to the described examples, and that othermethods of preparation could be suitable to prepare reagents accordingto the invention.

EXAMPLE 1 Preparation of Monopeptide Derivative of Rhodamine 110Employing the EDAC Procedure

A 10-fold excess of a FMOC amino acid is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (V:V) andstirred until complete solution occurs. To this stirred solution isadded a 12-fold excess of EDAC and the admixture is stirred for 30minutes. A solution of rhodamine 110 dissolved in a minimum of a 50:50pyridine-dimethylformamide (V:V) is added dropwise to the reactionsolution. This addition requires 15-20 minutes and the reaction solutionis allowed to stir at room temperature overnight. The solution isconcentrated under reduced pressure to an oil. This oil is dissolvedinto an appropriated organic solvent and the product is purified bynormal phase HPLC, using solvents of increasing polarity (methylenechloride, 1% methanol-chloroform, 2% methanol-chloroform, etc.). Theeluate containing the product is concentrated under reduced pressureaffording a crystalline material and the purity and identity are checkedby analytical reverse phase high pressure liquid chromatography and thinlayer chromatography.

The crystalline material is treated with a 5% solution of piperidine,dissolved in dimethylformamide. The reaction is stirred for 45 minutesand concentrated under reduced pressure. The resulting solid istriturated several times with pentane and then dissolved in a minimum ofmethanol and a 5-fold excess of trifluoroacetic acid is added. Thesolution is concentrated under reduced pressure to dryness and theresulting solid is centrifuged with cold diethyl ether until the ethertriturate has a pH=7. If the monopeptide is polar, then the remainingprotective group is removed by treating with a 30 to 50% trifluoroaceticacid solution in methylene chloride for four hours at room temperature.The solution is concentrated under reduced pressure to dryness and theresulting solid is centrifuged with cold diethyl ether until the ethertriturate has a pH=7. A final purification of this trifluoroacetic acidsubstrate is effected with reverse phase EPLC, using solvents ofdecreasing polarity (water, acetonitrile, trifluoroacetic acid). Theeluate containing the product is concentrated under reduced pressure andthe aqueous solution is lyophilized. The product's purity and identityare checked by analytical reverse phase high pressure liquidchromatography, thin layer chromatography, and photon countingspectrofluorometry. The purity and stability of the product are alsomeasured by monitoring the background fluorescence, autohydrolysis andenzymatic activity using the product as a substrate after storage of theproduct at 4° C. FIGS. 10A and 10B illustrate the stability and purityof a monopeptide-TFA salt derivative Proline-rhodamine 110 which wasprepared by the procedure described in this Example. Stability(background fluorescence) is shown in FIG. 10A. Autohydrolysis(diamonds) and enzyme rate (squares) are shown in FIG. 10B.

EXAMPLE 2 Preparation of Dipeptide Derivative of Rhodamine 110 Employingthe EDAC Procedure

A 6-fold excess of the FMOC amino acid is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (V:V) andstirred several minutes. To this well-stirred solution is added a12-fold excess of EDAC and the admixture is stirred an additional 30minutes. A solution of the monopeptide of rhodamine 110 dissolved in aminimum of 50:50 pyridine-dimethylformamide solution (V:V) is addeddropwise over a period of 15 to 20 minutes. The reaction is stirred atroom temperature for 16 hours and then concentrated to an oil underreduced pressure. This oil is dissolved in a minimum of an organicsolvent and the crude product is purified by normal phase HPLC. Theeluate containing the desired product is collected and concentratedunder reduced pressure affording a crystalline material and the purityand identity are checked by analytical reverse phase high pressureliquid chromatography and thin layer chromatography. The FMOC blockinggroup is removed by dissolving the solid in a 5%piperidine-dimethylformamide solution and stirred at room temperaturefor one hour. The solution is concentrated to dryness under reducedpressure. The resulting solid is triturated several times with pentaneto remove the FMOC polymer. The solid is dissolved in a minimum ofmethanol and a 5-fold excess of trifluoroacetic acid is added. Thesolution is concentrated to dryness and the resulting solid iscentrifuged with cold diethyl ether until the ether triturate has apH=7. If the dipeptide is polar, then the remaining protective group(s)is removed by treating with a 30 to 50% trifluoroacetic acid solution inmethylene chloride for four hours at room temperature. The solution isconcentrated under reduced pressure to dryness and the resulting solidis centrifuged with cold diethyl ether until the ether triturate has apH=7. A final purification of this trifluoroacetic acid substrate iseffected with reverse phase HPLC. The eluate containing the product isconcentrated under reduced pressure and the aqueous solution islyophilized. The product's purity and identity are checked by analyticalreverse phase high pressure liquid chromatography, thin layerchromatography and photon counting spectrofluorometry. The purity andstability of the product are also measured by monitoring the backgroundfluorescence, autohydrolysis and enzymatic activity using the product asa substrate after storage of the product at FIGS. 11A-11F illustrate thestability and purity of the TFA salts of several dipeptide derivativesof rhodamine 110 prepared by the procedure described in this Example.The Figures describe the following:

FIG. 11A (background fluorescence, Val-Lys•TFA);

FIG. 11B (autohydrolysis and enzyme rate, Val-Lys•TFA);

FIG. 11C (background fluorescence, Val-Ser•TFA);

FIG. 11D (autohydrolysis and enzyme rate, Val-Ser•TFA);

FIG. 11E (background fluorescence, Leu-Gly•TFA); and

FIG. 11F (autohydrolysis and enzyme rate, Leu-Gly•TFA).

EXAMPLE 3 Preparation of Polypeptide Derivative of Rhodamine 110Employing the EDAC Procedure

A 6-fold excess of the FMOC polyamino acid is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (V:V) andstirred until solution occurs. To this well stirred solution is added a12-fold excess of EDAC and the admixture is stirred an additional 30minutes. A solution of the monopeptide of rhodamine 110 dissolved in aminimum of 50:50 pyridine-dimethylformamide solution (V:V) is addeddropwise over a period of 15 to 20 minutes. The reaction is stirred atroom temperature for 16 hours and then concentrated to an oil underreduced pressure. The oil is dissolved in a minimum of an organicsolvent and the crude product is purified by normal phase HPLC. Theeluate containing the desired product is collected and concentratedunder reduced pressure affording a crystalline material and the purityand identity are checked by analytical reverse phase high pressureliquid chromatography and thin layer chromatography. The FMOC blockinggroup is removed by dissolving the solid in a 5% piperidinedimethylformamide solution and stirred at room temperature for one hour.The solution is concentrated under reduced pressure to dryness underreduced pressure. The resulting solid is triturated several times withpentane to remove the FMOC polymer. The solid is dissolved in a minimumof methanol and a 5-fold excess of trifluoroacetic acid is added. Thesolution is concentrated under reduced pressure to dryness and theresulting solid is centrifuged with cold diethyl ether until the ethertriturate has a pH=7. If the polypeptide is polar then the remaininggroup(s) is removed by treating with a 30 to 50% trifluoroacetic acidsolution for four hours at room temperature. The solution isconcentrated under reduced pressure to dryness, and the resulting solidis centrifuged with cold diethyl ether until the ether triturate has apH=7. A final purification of this trifluoroacetic acid substrate iseffected with reverse phase HPLC. The eluate containing the product isconcentrated under reduced pressure and the aqueous solution islyophilized. The product's purity and identity are checked by analyticalreverse phase high pressure liquid chromatography, thin layerchromatography and photon counting spectrofluorometry.

EXAMPLE 4 Preparation of a Dipeptide Derivative of Rhodemine 110Employing the HOBT-BOP Procedure

A 4-fold excess of the FMOC amino acid, and a 4-fold excess of EOBT andBOP are placed into a round bottom flask containing a 0.6 millimolarsolution of N-methylmorpholine in dimethylformamide and stirred for10-15 minutes. To this solution is added dropwise a solution of themonopeptide of rhodamine 110 dissolved in a minimum amount of a 0.6millimolar solution of N-methylmorpholine in dimethylformamide. Thisaddition requires 5-10 minutes, and the reaction is stirred at roomtemperature for four hours. The reaction solution is concentrated underreduced pressure to an oil. This oil is dissolved in methylene chlorideand the crude product is purified by normal phase HPLC. The eluatecontaining the desired product is collected and concentrated underreduced pressure affording a crystalline material. The purity andidentify of this material are checked by analytical reverse phase HPLCand thin layer chromatography. The FMOC blocking is removed bydissolving the solid in a 5% piperidine-dimethylformamide solution andstirred at room temperature for one hour. The solution is concentratedunder reduced pressure, and the resulting solid is triturated severaltimes with pentane to remove the FMOC polymer. The remaining solid isdissolved in a minimum of methanol and a 5-fold excess oftrifluoroacetic acid is added. The solution is concentrated underreduced pressure and the resulting solid is centrifuged with colddiethyl ether until the ether triturate has a pH=7. If the dipeptide ispolar then the remaining protective group(s) is removed by treating witha 30 to 50% trifluoroacetic acid solution in methylene chloride for fourhours at room temperature. The solution is concentrated under reducedpressure to dryness, and the resulting solid is centrifuged with colddiethyl ether until the ether triturate has a pH=7. A final purificationof this trifluoroacetic acid substrate is effected with reverse phaseHPLC. The eluate containing the product is concentrated under reducedpressure and the aqueous solution is lyophilized. The product's purityand identity are checked by analytical reverse phase high pressureliquid chromatography, thin layer chromatography and photon countingspectrofluorometry.

EXAMPLE 5 Preparation of a Polypeptide Derivative of Rhodamine 110Employing the HOBT-BOP Procedure

A 4-fold excess of the FMOC polypeptide and a 4-fold excess of HOBT andBOP are placed into a round bottom flask containing a 0.6 millimolarsolution of N-methylmorpholine in dimethylformamide and stirred for10-15 minutes. To this solution is added dropwise a solution of themonopeptide rhodamine 110 dissolved in a minimum amount of a 0.6millimolar solution of N-methylmorpholine in dimethylformamide. Thisaddition requires 5-10 minutes, and the reaction is stirred at roomtemperature for four hours. The reaction solution is concentrated underreduced pressure to an oil. This oil is dissolved in methylene chlorideand the crude product is purified by normal phase HPLC. The eluatecontaining the desired product is collected and concentrated underreduced pressure affording a crystalline material. The purity andidentify of this material are checked by analytical reverse phase HPLCand thin layer chromatography. The FMOC blocking is removed bydissolving the solid in a 5% piperidine-dimethylformamide solution andstirred at room temperature for one hour. The solution is concentratedunder reduced pressure, and the resulting solid is triturated severaltimes with pentane to remove the FMOC polymer. The remaining solid isdissolved in a minimum of methanol and a 5-fold excess oftrifluoroacetic acid is added. The solution is concentrated underreduced pressure and the resulting solid is centrifuged with colddiethyl ether until the ether triturate has a pH=7. If the polypeptideis polar then the remaining protective group(s) is removed by treatingwith a 30 to 50% trifluoroacetic acid solution in methylene chloride forfour hours at room temperature. The solution is concentrated underreduced pressure to dryness, and the resulting solid is centrifuged withcold diethyl ether until the ether triturate has a pH=7. A finalpurification of this trifluoroacetic acid substrate is effected withreverse phase HPLC. The eluate containing the product is concentratedunder reduced pressure and the aqueous solution is lyophilized. Theproduct's purity and identify are checked by analytical reverse phaseHPLC, thin layer chromatography and photon counting spectrofluorometry.

EXAMPLE 6 Preparation of p-Aminobenzoic Acid Derivative of Rhodamine 110

A molar quantity of p-Aminobenzoic Acid is placed into a round bottomflask containing a small quantity of dioxane and stirred until acomplete solution occurs. A 10% molar excess solution of sodiumcarbonate, dissolved in water, is added. To this well-stirred solutionis added dropwise a molar solution of9-fluorenylmethyloxycarbonylchloride dissolved in a minimum of dioxane.This addition requires 10 to 15 minutes and the reaction solution isallowed to stir an additional four (4) hours. The reaction is dilutedwith water and extracted (3) times with diethyl ether. The aqueous layeris cooled in an ice water bath and the pH is adjusted to two (2) with a10% solution of hydrochloric acid. The resulting colorless precipitateis filtered and recrystallized from an acetone solution. A TLC of thecolorless, crystalline product showed only one (1) quenched spot andobtained in a yield of 68%.

A 6-fold excess of the FMOC-p-amino acid is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (V:V) andstirred until a complete solution occurs. To this stirred solution isadded a 12-fold excess of EDAC and the admixture is stirred for 30minutes. A solution of rhodamine 110, dissolved in a minimum of a 50:50pyridine-dimethylformamide (V:V) is added dropwise to the reactionsolution. This addition requires 15-20 minutes, and the reactionsolution is concentrated under reduced pressure to an oil and dissolvedin a small amount of chloroform. The crude product is purified by normalphase HPLC and the product is eluted in a 5% methanol-chloroformsolution. This eluate is concentrated under reduced pressure, and theresulting colorless solid dried in vacuo affording a 60% yield of theproduct. This material is treated with a 5% solution of piperidinedissolved in dimethylformamide. The resulting solution is stirred atroom temperature for one (1) hour and concentrated under reducedpressure. The resulting solid is triturated several times with pentane,dissolved in a minimum of methanol and a 5-fold excess oftrifluoroacetic acid is added. The solution is concentrated to drynessand the resulting solid is centrifuged with cold diethyl ether until theether triturate has a pH of seven (7). The trifluoroacetate salt isdried in vacuo overnight affording a yield of 74.5%. The product'spurity and identify are checked by analytical reverse phase highpressure liquid chromatography, thin layer chromatography and photoncounting spectrofluorometry.

EXAMPLE 7 Preparation of Tetraacetyl-α-D-Glucopyranosyl Derivative ofRhodamine 110 and Tetrabenzoyl-α-D-Glucopyranosyl Derivative ofRhodamine 110

A 10-fold excess of the respective protected tetraacetyl (ortetrabenzoyl) α-D-glucopyranosyl bromides is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (V:V) andwarmed and stirred until a complete solution occurs. To this stirredsolution is added a 12-fold excess of EDAC and the admixture is stirredfor 30 minutes. A solution of rhodamine 110, dissolved in a minimum of a50:50 pyridine-dimethylformamide (V:V) is added dropwise to the reactionsolution. This addition requires 15 to 20 minutes. The reaction solutionis allowed to stir for 24 hours and concentrated under reduced pressureto an oil. The crude product is dissolved in chloroform and purified bynormal phase HPLC. The tetraacetyl-α-D-glucopyranosyl derivative iseluted in a 1% methanol-chloroform solution. Thetetrabenzoyl-α-D-glucopyranosyl derivative is eluted in a 3%methanol-chloroform solution. The product is isolated by concentratingthe respective eluates under reduced pressure and drying in vacuoovernight. The yield of the tetraacetyl-α-D-glucopyranosyl rhodamine 110is 30% and the yield of the tetrabenzoyl-α-D-glucopyranosyl rhodamine110 is almost quantitative (100%). The product's purity and identity arechecked by analytical reverse phase high pressure liquid chromatography,thin layer chromatography and photon counting spectrofluorometry.

EXAMPLE 8 Preparation of N-Butyl Ester Derivative of Fluorescein

A 3.4 fold excess of n-butyrylanhydride is placed into a round bottomflask containing a minimum amount of tetrahydrofuran and stirred forseveral minutes. A one molar equivalent of fluorescein is added,followed by 10 mL of triethylamine and solution occurs. The reactionsolution is stirred an additional 30 minutes and then concentrated underreduced pressure to an oil. The oil is dissolved in a minimum ofchloroform and the crude product is purified by normal phase HPLC. Theproduct is eluted with a 0.5% methanol-chloroform solution and theeluate is concentrated under reduced pressure affording a yield of acolorless solid of 63%. The product's purity and identity are checked byanalytical reverse phase high pressure liquid chromatography, thin layerchromatography and photon counting spectrofluorometry.

EXAMPLE 9 Preparation of Chloroacetyl Ester Derivative of Fluorescein

A 10-fold excess of chloroacetic anhydride is placed into a round bottomflask containing a minimum amount of tetrahydrofuran and stirred forseveral minutes. A solution of fluorescein, dissolved in a minimumamount of tetrahydrofuran and a 2-fold excess of triethylamine, is addeddropwise to the reaction mixture. This addition required 10 to 15minutes and the reaction solution is allowed to stir overnight. Thesolution is concentrated under reduced pressure to an oil. This oil isdissolved in a minimum amount of methylene chloride and the crudeproduct is purified by normal phase HPLC. The desired product is elutedin a 100% methylene chloride solution, and this eluate concentratedunder reduced pressure. This solid is dried in vacuo for 16 hoursaffording a quantitative yield (100%) of the desired product. Theproduct's purity and identity are checked by analytical reverse phasehigh pressure liquid chromatography, thin layer chromatography andphoton counting spectrofluorometry.

EXAMPLE 10 Preparation of n-Palmityl Ester Derivative of Fluorescein

A 2.5-fold excess of palmitic acid is placed into a round bottom flaskcontaining a minimum amount of tetrahydrofuran and stirred for severalminutes. To this solution is added a 3-fold excess of EDAC and themixture stirred for 30 minutes. A solution of fluorescein dissolved in aminimum amount of tetrahydrofuran is added dropwise to the reactionmixture. This addition required 15 to 20 minutes and the reactionmixture is allowed to stir overnight. The reaction mixture isconcentrated under reduced pressure to an oil. This oil is dissolved inchloroform and extracted three (3) times with a 5% aqueous sodiumbicarbonate solution. The organic layer is dried over magnesium sulfate,filtered and concentrated to dryness. This crude product is purified bynormal phase KPLC and the product is eluted with 100% chloroform. Thiseluate is concentrated under reduced pressure and the resulting solid isdried in vacuo affording 400 mg (9% yield). The product's purity andidentity are checked by analytical reverse phase high pressure liquidchromatography, thin layer chromatography and photon countingspectrofluorometry.

An elemental analysis for carbon and hydrogen, by GalbraithLaboratories, Inc. of Knoxville, Tenn., gave the following results:Formula: C₅₂ H₇₂ O₇. 1/2H₂ O MW=817,075

    ______________________________________                                        Theoretical           Found                                                   ______________________________________                                        C =       76.34       C =    76.36;                                                                              76.51                                      H =        8.99       H =     9.01;                                                                               9.05                                      ______________________________________                                    

EXAMPLE 11 Preparation of Diphenylphosphate Derivative of Rhodamine 110

A 6.6-fold excess of diphenylchlorophosphate is placed into a roundbottom flask containing a very small amount of pyridine and stirredseveral minutes in an ice-water bath. To this well-stirred, coldsolution is added rhodamine 110 and a white precipitate is formedimmediately. The stirring is continued for an hour and the reactionmixture is placed in the refrigerator for 48 hours. The reaction mixtureis treated with water and extracted twice with chloroform. The combinedchloroform extracts are dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product is purified bynormal phase HPLC. The desired product is eluted in a 1%methanol-chloroform solution, and this eluate is concentrated underreduced pressure to an oil. This oil is dissolved in ammonia, and theresulting aqueous solution is lyophilized affording the ammonium salt ofthe product in a 66% yield. The product's purity and identity arechecked by analytical reverse phase high pressure liquid chromatography,thin layer chromatography and photon counting spectrofluorometry.

EXAMPLE 12 Preparation of Diphenylphosphate Derivative of Fluorescein

A 6.6-fold excess of diphenylchlorophosphate is placed into a roundbottom flask containing a very small amount of pyridine and stirredseveral minutes in an ice-water bath. To this well-stirred, coldsolution is added fluorescein and a white precipitate is formedimmediately. Stirring is continued for one (1) hour and the reactionmixture placed in the refrigerator for 48 hours. The reaction mixture istreated with water and extracted twice with chloroform. The combinedchloroform extracts are dried over magnesium sulfate, filtered andconcentrated to dryness under reduced pressure. The resulting solid isdissolved in a minimum amount of chloroform and the crude product ispurified by normal phase HPLC. The product is eluted in a 1%methanol-chloroform solution and this solution concentrated underreduced pressure. The colorless solid is treated with ammonia andlyophilized, affording the ammonium salt in a yield of 95%. Theproduct's purity and identity are checked by analytical reverse phasehigh pressure liquid chromatography, thin layer chromatography andphoton counting spectrofluorometry.

EXAMPLE 13 Preparation of Trifluoroacetyl Ester Derivative of4'(5')Carboxyfluorescein

A 10-fold excess of trifluoroacetic anhydride is placed into a roundbottom flask containing a minimum amount of tetrahydrofuran and stirredseveral minutes. A 30% pyridine-tetrahydrofuran solution, containing the4'(5')carboxyfluorescein, is added dropwise over 10 to 15 minutes. Thesolution is allowed to stir overnight and concentrated under reducedpressure. The resulting oil is dissolved in chloroform, extracted threetimes with water and the organic layer dried over magnesium sulfate.This is filtered, concentrated to a small volume under reduced pressureand the crude product purified by normal phase HPLC. The product iseluted in a 4% methanol-chloroform solution. This is concentrated underreduced pressure and resulting solid dried in vacuo for 15 hoursaffording an 83% yield. The product's purity and identity are checked byanalytical reverse phase high pressure liquid chromatography, thin layerchromatography and photon counting spectrofluorometry.

EXAMPLE 14 Preparation of Diphenylphosphate Ester Derivative of4'(5')Carboxyfluorescein

A 15-fold excess of chlorodiphenylphosphate is added to a solution of4'(5')carboxyfluorescein, dissolved in 8 mL of pyridine over a period of10 to 15 minutes. The reddish-colored solution turns a light yellow anda precipitate is formed. Stirring is continued for two hours and themixture is allowed to cool in the refrigerator overnight. To thismixture is added 100 mL of water and the mixture extracted three timeswith chloroform. The combined chloroform extracts are dried overmagnesium sulfate, filtered and concentrated under reduced pressure toan oil. This oil is dissolved in a minimum amount of methylene chloride,and the crude product is purified by normal phase HPLC. The desiredproduct is eluted in a 1% methanol-chloroform solution, and this eluateis concentrated under reduced pressure. The resulting solid is dried invacuo for 16 hours affording a yield of 95%. The product's purity andidentity are checked by analytical reverse phase high pressure liquidchromatography, thin layer chromatography and photon countingspectrofluorometry.

EXAMPLE 15 Preparation of H-L-Leucine Trifluoroacetate Salt Derivativeof Rhodol

A 10-fold excess of benzyloxycarbonyl-L-leucine is placed into a roundbottom flask containing a 50:50 pyridine-dimethylformamide solution(V:V) and stirred until a complete solution occurs. To this stirredsolution is added a 12-fold excess of EDAC and the admixture is stirredfor 30 minutes. A solution of rhodol hydrochloride, dissolved in aminimum of a pyridine-dimethylformamide solution (V:V), is addeddropwise to the reaction solution. This addition required 10 to 15minutes and the reaction is allowed to stir at room temperatureovernight. The solution is concentrated under reduced pressure to anoil. This oil is dissolved into chloroform and extracted three (3) timeswith water and organic layer dried over magnesium sulfate. This isfiltered, concentrated to a-very small volume under reduced pressure andpurified by normal phase HPLC. The product is eluted in a 2%methanol-chloroform solution. This eluate is concentrated under reducedpressure and the resulting colorless, crystalline solid, dried in vacuo,affords a 33.4% yield of the product and the product's purity andidentity are checked by analytical reverse phase high pressure liquidchromatography and thin layer chromatography. This material is dissolvedinto a small volume of isopropyl alcohol and catalytically reduced witha small amount of 10% palladium on carbon as the catalyst in a Paarshaker apparatus for 16 hours. The alcohol solution is carefullyfiltered and a 2-fold excess of trifluoroacetic acid is added. Thissolution is concentrated to dryness under reduced pressure and theresulting solid is centrifuged with cold diethyl ether until the ethertriturate has a pH of 7. The colorless trifluoroacetate salt is dried invacuo overnight, affording a 92.48% yield. The product's purity andidentity are checked by analytical reverse phase high pressure liquidchromatography, thin layer chromatography and photon countingspectrofluorometry.

EXAMPLE 16 Preparation of (H-LEU-GLY)₂ Rhodamine 110 Acetate andTartrate Salts

A 10-fold excess of FMOC glycine is placed into a round bottom flaskcontaining a 50:50 pyridine-dimethylformamide solution (v:v) and stirreduntil a complete solution occurs. To this stirred solution is added a12-fold excess of EDAC and the admixture is stirred for 30 minutes. Asolution of rhodamine 110 is dissolved in a minimum of a 50:50pyridine-dimethylformamide solution (v:v) and is added dropwise to thereaction solution. This addition requires 15-20 minutes, and thereaction is allowed to stir at room temperature overnight. The solutionis concentrated under reduced pressure to an oil. This oil is dissolvedin a small amount of methylene chloride, and the product is purified bynormal phase EPLC. The product is eluted from the column in a 1%methanol-chloroform solution. This eluate is concentrated under reducedpressure, and the resulting solid dried in vacuo affording an 85% yieldof the product. The purity and identity are checked by analyticalreverse phase high pressure liquid chromatography and thin layerchromatography. This material is treated with a 5% solution ofpiperidine dissolved in dimethylformamide. The resulting solution isstirred at room temperature for one (1) hour and concentrated underreduced pressure. The resulting solid is triturated several times withpentane and product dried in vacuo. A TLC of this material showed onlyone quenched spot which is positive to concentrated hydrochloric acid.

A four (4) fold excess of FMOC-L-leucine is placed into a round bottomflask containing a 50:50 pyridine-dimethylformamide solution (v:v) andstirred until a complete solution occurs. To this stirred solution isadded an eight (8) fold excess of EDAC and the admixture is stirred for30 minutes. A solution of (H-GLY)₂ rhodamine 110 (from above) dissolvedin a minimum of a 50:50 pyridine-dimethylformamide solution (v:v) isadded dropwise to the reaction solution. This addition requires 15-20minutes, and the reaction is allowed to stir at room temperature for six(6) hours. The solution is concentrated under reduced pressure to anoil. This oil is dissolved in chloroform, and the product is purified bynormal phase high pressure liquid chromatography. The product is elutedfrom the column in a 2% methanol-chloroform solution. This eluate isconcentrated under reduced pressure, and the resulting solid dried invacuo affording a 74% yield. The purity and identity are checked byanalytical reverse phase high pressure liquid chromatography and thinlayer chromatography. This material is treated with a 5% solution ofpiperidine dissolved in dimethylformamide. The resulting solution isstirred at room temperature for one (1) hour and concentrated underreduced pressure. The solid is triturated several times with pentane anddried in vacuo. One half of this material is dissolved in a small amountof methanol and a 10% excess of acetic acid is added. Ether is added tothis solution and cooled in an ice-water bath. The resulting colorlesssolid is filtered, washed with ether and centrifuged with ether untilthe pH=7. The crystalline salt is dried in vacuo according a 61% yield.

The remaining one-half of the material (from above) is dissolved in asmall amount of methanol and a 10% excess of L-tartaric acid dissolvedin a very small amount of methanol is added. This solution is cooled inan ice-water bath and ether is added. The resulting crystalline materialis filtered, washed with ether and centrifuged with ether until thepH=7. The resulting, colorless salt is dried in vacuo affording a 42%yield. The free fluorescence and identity of the acetate and tartratesalts of these rhodamine 110 substrates are checked by analyticalreverse phase high pressure liquid chromatography, thin layerchromatography and photon counting spectrofluorometry.

The acetate and tartrate salts thus prepared have the followingcharacteristics, respectively: native free fluorescence, 63,000 and61,000 photons; autohydrolysis rate when measured at 37° C. using a 1 cmpath length, -5.56 and -3.8 change in photons per second; and enzymaticreaction rate of cathepsin B at 37° C., +128 and +138 change in photonsper second. The purity and stability of acetate and tartrate salts of(LeuGly)₂ rhodamine 110 as demonstrated by assessment of autohydrolysis,background fluorescence and enzymatic activity after storage at 4° C.are illustrated in FIGS. 12A-12D as follows:

FIG. 12A (background fluorescence, Leu-Gly•acetate);

FIG. 12B (autohydrolysis and enzyme rate, Leu-Gly•acetate);

FIG. 12C (background fluorescence, Leu-Gly•tartrate); and

FIG. 12D (autohydrolysis and enzyme rate, Leu-Gly•tartrate).

EXAMPLE 17 Preparation of the Free Amine of (Lys-Ala)₂ Rhodamine 110

A 10-fold excess of the FMOC L-lysine εBOC amino acid is placed into around bottom flask containing a 50:50 pyridine-dimethylformamidesolution (V:V) and stirred several minutes. To this well-stirredsolution is added a 20-fold excess of EDAC and the admixture is stirredan additional 30 minutes. A solution of (Ala)₂ rhodamine 110 dissolvedin a minimum of 50:50 pyridine-dimethylformamide solution (V:V) is addeddropwise over a period of 15 to 20 minutes. The reaction is stirred atroom temperature for 16 hours and then concentrated to an oil underreduced pressure. This oil is dissolved in a minimum of an organicsolvent and the crude product is purified by normal phase HPLC. Theeluate containing the desired product is collected and concentratedunder reduced pressure affording a crystalline material. A TLC of thismaterial is run to check for purity and identity. The BOC protectinggroup is removed by dissolving the solid into a 50% solution oftrifluoroacetic acid in methylene chloride. The reaction is stirred atroom temperature for one hour, and the purity of the reaction product ischecked by thin layer chromatography. The TLC did not show any of theBOC group. The acid solution is concentrated under reduced pressure todryness. Several washes with fresh methylene chloride andreconcentrations under reduced pressure are performed to generate acrystalline solid. The FMOC blocking group is removed by dissolving thesolid in a 5% piperidine-dimethylformamide solution and stirred at roomtemperature for one hour. The solution is concentrated to dryness underreduced pressure. The resulting solid is triturated several times withpentane to remove the FMOC polymer and the product is dried in vacuo toconstant weight affording a yield of 98.62%. The purity of this materialis checked by reverse phase HPLC, thin-layer chromatography and photoncounting spectrofluorometry. The stability and purity of the product isfurther determined by monitoring the autohydrolysis, backgroundfluorescence and enzymatic activity with the product as a substrateafter storage of the product at 4° C. FIGS. 13A and 13B illustrate thestability of the free amine of (Lys-Ala)₂ rhodamine 110 which wasprepared by the procedure described in this Example. FIG. 13A showsbackground fluorescence and FIG. 13B shows autohydrolysis and enzymerate.

EXAMPLE 18 Use of Different Salts to Enhance Specificity

The use of salts to identify cellular enzymes is very important. pHoptimas are different demonstrating different enzymes or isoenzymes.Different salts from within the same pH range may give differentreactivities. Z-groups, which are not salts but covalent organiccompounds, show relatively little activity and no pH optima. See FIGS.2A-2D, using cathepsin B as a target enzyme.

EXAMPLE 19 Use of Inhibitors in the Reagent Formula

Use of inhibitors of the targeted enzyme has been shown to provesubstrate specificity. More specifically, when an inhibitor eliminatedthe targeted enzyme signal, it was reasoned that the targeted enzymeactivity was measured without the inhibitor. The disclosed enzyme assaycontemplates the use of interfering reaction inhibitors to increase andmaintain specificity.

To improve a Cathepsin D response, inhibitors to aminopeptidase andCathepsin B are added to the substrate most specific for Cathepsin D.Conversely, adding a Cathepsin D inhibitor to an assay for Cathepsin Drequires measurement before and after inhibitor addition thus requiringtwo (2) measurements per assay. The opposite approach only requires onemeasurement. See FIGS. 3A-3D.

EXAMPLE 20 Immune Competence

The cell's ability to fight off an invader lies within its genetics andtherefore cell type. The "readiness" however of any genetically capablegroup of cells to defend is different. A measure of this "readiness" ismanifest in the available proteolytic enzymes contained within vacuolesor on the surface of the cell. The assay compound hydrolysis rateincreases with increased mass of enzymes giving a picture of immunecompetence both in number of cells and activity level. FIG. 4A showscell size (fs), granularity (ss) and amino peptidase activity (logfluorescence at 525 nm v. time) using Leu rhodamine 110-TFA as asubstrate in normal Ficoll prepared lymphocytes. FIG. 4B shows the samedata for acute lympholytic Ficoll prepared lymphocytes. The cells testedin FIG. 4B have lost their enzymatic activity. Images were generatedusing Universal Imaging.

EXAMPLE 21 Leukemia

A panel of assay compounds are assembled consisting ofpro-aminopeptidase; aminopeptidase M (Pro, Lys, Gly, Ala, Leu),Cathepsin D (Gly-Leu, Thr-Pro), Cathepsin B (Gln-Ser, Leu-Gly, Val-Ser,Val-Lys), Cathepsin C (Ala-Gly) and dipeptidyl peptidase II (Lys-Ala,Gly-Pro, Ala-Ala). Values for these assay compounds outside the normalrange are considered diagnostic for leukemia. In addition, the ratios ofthese enzyme readings to one another provide information on furtherclassifying the leukemia into myelogenous or lymphocytic and monitoringthe course of the disease. Values may be both higher or lower than thenormal range. FIG. 5A shows results obtained when normal leukocytes aretested with various rhodamine 110-monopeptide and rhodamine110-dipeptide compounds. All compounds except (Lys-Ala)₂ rhodamine 110are TFA salts. (Lys-Ala)₂ rhodamine 110 was a free amine derivative.FIG. 5B shows results obtained when leukemia cells are tested withvarious rhodamine 110-monopeptide compounds.

EXAMPLE 22 Sepsis

A panel of assay compounds are assembled consisting of aminopeptidase(Leu, Pro, Lys, Gly, Ala), dipeptidyl peptidase II (Gly-Pro, Lys-ala,Ala-Ala), Cathepsin C (Ala-Gly) and Cathepsin B (Leu-Gly, Val-Lys,Val-Ser and Gln-Ser) and cathepsin D (Gly-Leu and Thr-Pro). Values forthese substrates outside the normal range are considered diagnostic forsepsis. FIG. 6A shows results obtained when cells from a patient thathad been shot by a gun and who was experiencing sepsis were treated withvarious rhodamine 110-monopeptide and dipeptide compounds. FIG. 6B showsresults obtained when umbilical cord blood cells from a newborn infantwere treated with various rhodamine 110-monopeptide and dipeptidecompounds. All compounds except (Lys-Ala)₂ rhodamine 110 are TFA salts.(Lys-Ala)₂ rhodamine 110 was a free amine derivative.

EXAMPLE 23 TB Infection

A panel of assay compounds are assembled consisting ofAla-aminopeptidase and Lys-aminopeptidase, Dipeptidyl peptidase IV(Ata-Ala)₂ rhodamine 110 and Cathepsin D to indicate possible TBinfection in AIDS related cases.

A panel of enzymatic substrates is performed consisting ofAla-aminopeptidase and Lys-aminopeptidase, Dipeptidyl peptidase IV(Ala-Ala)₂ rhodamine 110 and Cathepsin D to indicate possible TBinfection in AIDS related cases. The results are reported in Table 4below:

                  TABLE 4                                                         ______________________________________                                                  NORMALS      HIV + PATIENTS                                                   MEAN               MEAN                                                       DELTA              DELTA           P                                SUBSTRATE FL      SD     N   FL    SD   N    2 TAIL                           ______________________________________                                        GLN-SER****                                                                             11.9    4.4    14  13.7  7.2  13   N.S.                             GLN-SER*  6.1     2.6    14  12.7  12.9 17   <0.060                           VAL-SER****                                                                             24.6    5.3    14  32.5  14.7 7    N.S.                             LYS-ALA.sup.1                                                                           112.6   43.5   12  121.5 62.9 7    N.S.                             LYS-ALA.sup.2                                                                           7.6     1.7    12  7.9   3.7  13   N.S.                             THR-PRO****                                                                             295.0   140.9  7   204.4 106.1                                                                              6    N.S.                             ALA-GLY****                                                                             48.0    44.1   11  39.9  43.7 6    N.S.                             ALA-GLY*  31.6    27.0   13  29.7  24.8 13   N.S.                             THR-PRO****                                                                             87.2    95.8   13  62.9  99.2 13   N.S.                             THR-PRO*  17.3    18.5   13  10.2  8.7  17   N.S.                             GLY-PRO****                                                                             46.7    42.8   17  18.4  9.2  13   <0.020                           GLY-PRO*  50.9    45.9   17  21.4  15.5 19   <0.025                           ALA-ALA****                                                                             158.4   47.5   9   110.1 31.6 16   <0.020                           ALA-ALA*  25.6    7.6    13  26.3  10.4 13   N.S.                             GLY-LEU****                                                                             9.5     4.7    17  16.3  11.1 17   <0.030                           LEU-GLY** 143.0   132.7  13  104.1 98.4 13   N.S.                             LEU-GLY***                                                                              162.4   146.9  13  111.9 99.8 13   N.S.                             VAL-LYS****                                                                             22.7    16.1   15  17.7  10.4 13   N.S.                             GLYCINE****                                                                             101.1   101.2  11  62.4  73.3 13   N.S.                             ALANINE****                                                                             132.0   140.4  13  59.0  31.3 17   <0.095                           LYSINE****                                                                              10.5    3.7    12  7.4   3.8  17   <0.040                           PROLINE****                                                                             11.4    4.3    10  11.0  5.0  13   N.S.                             LEUCINE****                                                                             259.6   99.6   8   130.5 99.5 9    <0.020                           ______________________________________                                         *BESTATIN + TFA RHODAMINE 110                                                 **TARTRATE                                                                    ***ACETATE                                                                    ****TFARHODAMINE 110                                                          .sup.1 Rho 110free amine                                                      .sup.2 Rho 110free amine + bestatin                                      

EXAMPLE 24 Metastatic Potential in Solid Tumors

A panel of assay compounds are assembled consisting of Cathepsin Bmarkers (Gln-Ser, Val-Ser), Cathepsin C (Thr-Pro), Dipeptidyl peptidaseIV (Ala-Ala) and Leu-aminopeptidase to predict metastatic potential insolid tumors. The results obtained when breast tumor cells (and onenormal breast control cell sample) are treated with TFA salts of variousrhodamine 110-peptide compounds are shown in FIG. 7.

EXAMPLE 25 Monitoring Drug Treatment

An assay compound can be used to monitor drug treatment. Enzymaticactivity according to drug target, i.e., protein synthesis, can diminishover time and increase dramatically depending on dose of drug. Theresults obtained when Raji cells which had been exposed to variousconcentrations of cyclophosphamide for 48 hours are treated withLeu-rhodamine 110 for 1 minute are shown in FIG. 8A. The resultsobtained when Raji cells, which had been exposed to variousconcentrations of vincristine for 48 hours, are treated with a TFA saltof Leu-rhodamine 110 for 1 minute and aminopeptidase activity ismeasured are shown in FIG. 8B.

EXAMPLE 26 Macrophage Activation

FIG. 9 illustrates the use of assays to provide an indication ofmacrophage activation. Using a mouse model, various eypes of cells usedto study metastatic versus non-metastatic breast tumors, were treatedwith Leu-rhodamine 110 substrate and amino peptidase activity wasmeasured. The results obtained are shown in FIG. 9.

EXAMPLE 27 Red Blood Cell Adenosine Deaminase (ADA) and Relationship ToHereditary Non-Spherocytic Hemolytic Anemia (HNSHA) Disease

Hereditary deficiencies of glycolytic enzymes or related pathways in theerythrocyte are characterized by the disease hemolytic anemia.Hereditary Non-Spherocytic Hemolytic Anemia is distinguished fromHereditary Spherocytosis by the fact that red blood cells aremorphologically normal and manifest a normal osmotic fragility. Only inthe case of pyrimidine 5' nucleotidase deficiency is the erythrocytemorphology changed to a basophilic stippling.

Deficiencies of ADA are well-known causes of immunodeficiency. In caseswhere ADA is greatly increased to levels as high as 100 times normal butother tissues have normal levels in the same individual, the clinicaldisease is HNSHA. The high ADA depletes the erythrocytes of vitaladenine nucleotides, impairing their metabolism. The residual enzymestructure is normal and the gene is normal but attaching the promoter toa reporter gene produced increased levels of enzyme.

The structure of Adenosine is: ##STR2##

The enzyme adenosine deaminase removes the NH₂ and replaces it with ahydroxyl: ##STR3##

The assay compound using rhodamine 110 is then: ##STR4##

Hydrolysis by ADA leaves 2 Inosine and 1 rhodamine 110: ##STR5##

To assay vital cells for ADA activity, a blood sample (containingplatelets, erythrocytes and leukocytes) is washed to remove plasma,debris, dead cells and extra cellular enzymes. The sample is incubatedat 37° C. in the wash media.

A media is prepared for the assay reagent using Hanks balanced salts atpH 7.0. The aqueous buffer media is adjusted to isosmotic conditions.The ionic strength is adjusted to 0.1 to 0.3μ by additions of salts.Appropriate cofactors including divalent cations such as Ca²⁺, Mg²⁺ andBa²⁺ are added for ADA to maximize the hydrolysis rate. The assaycompound is added at excess for the quantities of enzyme analyzed. (Atime-course activity assay is used to determine correct fluorescenceintensity data collection, usually between 10 seconds and 10 minutes, aswell as, appropriate assay compound concentration.)

The washed, pre-incubated blood sample is added to the media, incubatedat 37° C. and fluorescent intensity is measured at the predeterminedtime on erythrocytes. The fluorescence found on platelets and leukocytesare disregarded. Separation of cell types is aided by sizediscrimination.

Comparison of "normal" erythrocytic ADA activity to those in HNSHAdisease state demonstrates a 100-fold increase in ADA activity.

The structure of pyrimidine 5'nucleotide is: ##STR6##

The enzyme pyrimidine 5'nucleotidase removes the phosphate group fromthe compound: ##STR7##

The assay reagent using fluorescein is then: ##STR8##

To assay vital cells for pyrimidine-5'-nucleotidase, a blood samplecontaining platelets, erythrocytes, leukocytes and plasma is washed toremove plasma, debris, dead cells and extracellular enzymes. The sampleis pre-incubated at 37° C. in the wash media.

A media is prepared for the assay reagent using glycine-sodium hydroxidebuffer at pH 8.5. The aqueous buffer media is adjusted to isosmoticconditions. The ionic strength is adjusted to 0.1 to 0.3μ by addition ofsalts. Appropriate cofactors of calcium chloride and magnesium sulfateare added for pyrimidine 5'nucleotidase to maximize the hydrolysis rate.The assay compound is added at excess for the quantities of enzyme to beanalyzed. Other Michaelis-Menten parameters are determined to providecorrect data collection window.

The washed pre-incubated blood sample is added to the media, incubatedat 37° C. and fluorescent intensity is measured on erythrocytes.Erythrocytes are identified visually under a microscope usingmorphological indicators. Erythrocytes are identified using a flowcytometer by size and granularity discrimination or 2-color assaymonoclonal antibody for erythrocytes.

Comparison of "normal" erythrocytes pyrimidine 5'nucleotidase to thosein HNSHA shows a deficiency of enzyme in HNSHA.

EXAMPLE 28 Proinsulin or Pre-proinsulin Inside Cell

Insulin is synthesized as a single chain polypeptide--pre-proinsulin.The signal sequence "pre" becomes cleaved during synthesis on the roughendoplasmic reticulum, and no mutations are known that causedisturbances of removing signal sequences, because such mutationsprobably would be lethal.

Proinsulin is characterized by the presence of a C-peptide that joinsthe two A and B chains of the mature insulin molecule. Mutations occurmostly at the two critical junctions where the C-peptide is attached tothe A and B insulin chains by two pairs of basic amino acids. Suchdefects have been recognized in families with hyperinsulinemia. Thedefect is inherited in an autosomal dominant pattern, and probablyinvolves the loss of one of the basic amino acid residues that makes itimpossible to cleave the proinsulin molecule at the mutation site, whichresults in the presence of a two-chained intermediate of proinsulinmolecules secreted into the blood plasma.

Clinically occurring glucose intolerance with abnormally high ratios ofproinsulin-like material to insulin (9 to 10 as compared with normalvalues of approximately 0.25) are due to a loss of Arg 65 and loss ofthe protective activity causing acetylation of Lys 64 which make itimpossible to cleave the C-peptide from the A chain of insulin. It issupposed that substitution of Arg 65 of the proinsulin molecule resultsin failure of cellular enzymes to cleave correctly the C-peptide fromthe A chain of insulin. Use of a specific substrate, designed for thiscleavage site, can be diagnostic for the defect, as well as prognosticfor genetic treatment.

EXAMPLE 29 Hairy Cell Leukemia

Acid-tartaric buffer in phosphate substrate is used to confirm hairycell leukemia. "Tartaric Resistant Acid Phosphatase" is a cellularcomponent found in hairy cell leukemia. Design of a buffer system, usingtartrate in the buffer and a phosphate substrate, confirms presence ofthis disease with a positive result.

EXAMPLE 30 Cathepsin B

Cathepsin B substrates Gln-Ser, Val-Ser, Leu-Gly, Val-Lys specific forisoenzymes of Cathepsin B. The use of isoelectric focusing for CathepsinB enzyme and nitrocellulose transfer of these isoenzymes, similar toWestern blot, provides a solid support to test substrate activity.Examination of substrate activity, based on design of the dye molecule,determines isoenzyme specificity based on structure.

To prepare the reagent for measuring cathepsin B activity, an assaycompound such as Val-Lys-rhodamine 110-TFA is dissolved in 100% DMSO ata stock concentration of 1.6 mM. The stock solution is then diluted20-fold with 10 mM MES buffer at pH 6.0! to give a reagent concentrationof 0.08 mM. To the 0.08 mM solution the following reagents are added.0.5 mM Bestatin as an aminopeptidase inhibitor, 1.0 mM dithiothretol asactivator, 1.0 mM CaCl₂ and 1.0 MgCl₂ as cofactors and 247 mM Mannitolas a bulking reagent for lyophilization. The complete reagent mixture islyophilized during which process the DMSO is effectively removed and thelyophilized mixture is reconstituted using endotoxin-free deionizedwater.

EXAMPLE 31 Response to Modulators

The use of cellular response modifiers, i.e., PMA (phorbol myristateacetate), interleukins and interferons in the pre-incubation stepprovides information on cellular response. If the cell function isnormal the response to the modulator will, for selected enzymesubstrates, be in defined ranges. If abnormal, the measured responsewill be higher or lower.

To make the reagent, the assay compound dichlorofluoroscein diacetate isdissolved in 100% DMSO at 6.0 mM as a stock solution. The stock solutionis diluted 100-fold with 10 mM MES at pH 6.0 to give a reagentconcentration of 0.06 mM. 0.032 mM PMA is added as a cell-activator and247 mM Mannitol is added as a bulking agent for lyophilization. Thereagent mixture is lyophilized which effectively removes the DMSO andreconstituted with endotoxin-free deionized water.

EXAMPLE 32 Use in Conjunction with Genetic Analysis Techniques

The methods to determine the activity of an enzyme using the assaysdescribed above are also useful when used in independent combinationwith genetic analysis techniques including, polymerase chain reaction(PCR), transcription mediated amplification (TMA), ligase chain reaction(LCR) and fluorescent in situ hybridization (FISH). The results obtainedusing these genetic analysis techniques can be used both forconfirmation of diagnostic conclusions based on measurements ofenzymatic activities in cells, as determined with the assays previouslydescribed herein, and for the differentiation of purely functionalpathologies and functional pathologies having an underlying geneticcause.

EXAMPLE 33 Statistical Analysis and Diagnosis of Normal and DiseasedStates based on Cellular Enzymatic Activity

A normal in house adult donor pool was drawn, recording sex anddetermining that no known disease state was present at the time ofsample draw. Criteria for rejecting samples included patients undermedication, patients with infection or inflammation of any type, coldsand flu as well as any known medical illness such as cancer, heartailment and high blood pressure. A donor list of 75 patients has beendeveloped and a mean and 2 sd range established. A subgroup of "supernormals" was developed from this list by examining the 75 patients todetermine which group falls closest to the mean for all enzymes in allcell types tested. These patients were used as the "wellness index" orbest group of "normalcy" patients. If a study required the use ofchildren or newborns then this same format was used to develop a database for these age groups. It was noted that the normal range wasdifferent in the different age groups. These groups were also screenedfor criteria, for example in the newborns an apgarde value greater than8, with a normal delivery, 48 hour stay in the hospital and no clinicaldiseases diagnosed in the mother was considered normal.

The next task was to develop a similar data base for different diseasestates. These were clinically diagnosed using conventional technology toidentify the disease. The patient samples were assayed using the sameprotocol as the normal samples. The patients were not transfused and thesample was less than 4 hours old at analysis. All cell type informationwas collected with all enzyme assays. Staging of the disease was notedwhere available and all drug treatments were also recorded. Drugpharmokinetics were determined by the appropriate reference to estimatethe drug's possible influence on cellular function. Separate studiesusing the drugs on human tissue culture cell lines were also used todetermine their effect on specific cell types and provide reference.Untreated patient samples were separated into their own group andcompared to drug treatment protocols. The goal of the studies was earlyprediction of disease states as well as monitoring of treatmentmodalities.

The data from the collected samples was organized into tables containingrows of cell type and enzyme concentration. As illustrated in Table 5,the first seven columns contain either individual patients with adisease or the mean of patients with a disease. Calculation of the sumof the squares, the covariance or the correlation of the rows thenindicated which enzyme and what cell type gave the largest differenceand therefore the most descriptive indicator of the disease. Thisapproach used the Non-Negative Least Squares method and the ANOVAmethod. Eigenvectors were then determined for an unknown to predict themost likely disease for the unknown. FIG. 16 shows the prediction of anunknown (actually diagnosed as JRA) from a group of inflammatorydiseases (Kawasaki #1, Lupus #2, Juvenile Rheumatoid Arthritis #3,Dermatomyositis #4, Rheumatic Fever #5, and Inflammatory Bowel Disease#6) using the full data matrix versus reduced data matrices usingrespectively: the enzyme/cell type combinations identified byeigenvector 1 alone, eigenvector 1 and 2 together, or the analysis basedon squared deviations from the mean (variance) alone.

                                      TABLE 5                                     __________________________________________________________________________    LYMPHOCYTES                                                                           Kawasaki's                                                                           Lupus                                                                             JRA         Rheumatic                                                                          Inflam.                                           Ratio  Ratio                                                                             Ratio                                                                             Dermatomyositis                                                                       Fever                                                                              Bowel Dis.                                                                          Eigen                                                                              Eigen     Inflam Dis           SUBSTRATE                                                                             Dis. Lymphs.                                                                         Lymphs                                                                            Lymphs                                                                            Ratio Lymphs                                                                          Lymphs                                                                             Lymphs                                                                              Vector 1                                                                           Vector 2                                                                           Variance                                                                           Lymphs               __________________________________________________________________________    LEU     0.93   1.93                                                                              1.64                                                                              1.36    1.05 1.34                 0.72                 ALA     1.05   1.16                                                                              1.59                                                                              1.16    1.18 1.53                 1.07                 GLY     1.94   1.64                                                                              0.17                                                                              1.30    2.09 1.97                 0.16                 VK      1.19   1.25                                                                              1.60                                                                              1.39    0.74 1.08                 1.29                 VK-M    0.83   0.99                                                                              7.71                                                                              0.97    0.69 0.56       3    5    4.64                 KA      1.53   1.93                                                                              0.10                                                                              1.39    1.19 1.75                 0.07                 KA-M    1.32   1.06                                                                              1.46                                                                              1.93    2.07 1.63                 2.06                 Z-GP    2.90   1.93                                                                              1.29                                                                              1.73    3.23 1.73                 1.07                 Z-GP-M  3.44   2.09                                                                              1.30                                                                              3.45    4.09 3.80                 1.04                 Z-TP 6.5                                                                              5.85   2.30                                                                              2.89                                                                              3.87    7.19 3.03                 1.71                 FDA     0.28   1.01                                                                              1.17                                                                              1.06    0.90 0.87                 0.80                 FDA-NAF 0.30   0.74                                                                              0.21                                                                              1.14    0.97 0.89                 0.16                 DCHFMESPMA                                                                            0.09   0.63                                                                              0.38                                                                              0.52    0.34 0.40                 0.31                 GFGA    2.15   1.25                                                                              2.03                                                                              2.14    2.14 2.16                 1.40                 RGES    1.43   1.84                                                                              1.43                                                                              2.06    2.04 1.96                 1.23                 DGLUC   0.25   0.78                                                                              0.82                                                                              0.72    0.97 1.04                 0.45                 DPO4    0.62   0.90                                                                              0.82                                                                              0.64    0.77 0.87                 0.45                 GALAC   2.46   1.30                                                                              2.28                                                                              2.34    2.24 2.39                 1.40                 __________________________________________________________________________    MONOCYTES                                                                                    Lupus                                                                             JRA         Rheumatic                                                                          Inflam.                                           Kawasaki's                                                                           Ratio                                                                             Ratio                                                                             Dermatomyositis                                                                       Fever                                                                              Bowel Dis.                                                                          Eigen                                                                              Eigen     Inflam. Dis.         SUBSTRATE                                                                             Dis. Monos                                                                           Monos                                                                             Monos                                                                             Ratio Monos                                                                           Monos                                                                              Lymphs                                                                              Vector 1                                                                           Vector 2                                                                           Variance                                                                           Monos                __________________________________________________________________________    LEU     0.57   44.44                                                                             1.44                                                                              1.35    0.96 1.13  1         1    0.65                 ALA     1.36   6.17                                                                              1.31                                                                              0.99    1.08 1.43  5              0.94                 GLY     3.83   6.00                                                                              1.11                                                                              1.01    2.06 1.84  6              0.35                 VK      5.38   1.64                                                                              5.94                                                                              1.42    1.32 3.08       7         2.32                 VK-M    0.25   11.37                                                                             3.49                                                                              3.45    8.67 1.41  2    6    3    1.87                 KA      2.22   6.93                                                                              0.54                                                                              1.04    2.39 1.95  4              0.25                 KA-M    7.08   14.08                                                                             12.14                                                                             1.55    15.36                                                                              4.10  3    1    2    1.44                 Z-GP    4.06   1.88                                                                              1.34                                                                              1.50    3.56 1.64                 0.98                 Z-GP-M  4.47   1.92                                                                              1.79                                                                              3.05    2.99 3.33                 0.73                 Z-TP 6.5                                                                              6.40   2.29                                                                              8.33                                                                              3.12    5.37 2.83       4         3.06                 FDA     0.41   1.01                                                                              1.55                                                                              0.94    0.63 0.98                 0.47                 FDA-NAF 0.58   0.83                                                                              0.35                                                                              1.03    0.89 1.11                 0.13                 DCHFMESPMA                                                                            0.13   0.48                                                                              0.38                                                                              0.47    0.35 0.46                 0.24                 GFGA    1.77   2.18                                                                              2.29                                                                              2.01    1.36 3.42                 1.67                 RGES    4.65   3.03                                                                              3.98                                                                              5.22    6.52 6.02                 0.61                 DGLUC   0.35   0.38                                                                              1.13                                                                              0.73    2.91 4.62                 0.28                 DPO4    0.51   0.69                                                                              1.13                                                                              0.79    1.09 1.30                 0.28                 GALAC   8.40   1.17                                                                              3.81                                                                              2.14    1.80 4.93            6    1.87                 __________________________________________________________________________    GRANULOCYTES                                                                                 Lupus                                                                             JRA         Rheumatic                                                                          Inflam.                                           Kawasaki's                                                                           Ratio                                                                             Ratio                                                                             Dermatomyositis                                                                       Fever                                                                              Bowel Dis.                                                                          Eigen                                                                              Eigen     Inflam. Dis.         SUBSTRATE                                                                             Dis. Grans                                                                           Grans                                                                             Grans                                                                             Ratio Grans                                                                           Grans                                                                              Lymphs                                                                              Vector 1                                                                           Vector 2                                                                           Variance                                                                           Grans                __________________________________________________________________________    LEU     0.33   2.03                                                                              1.43                                                                              1.31    0.98 1.11                 0.58                 ALA     1.31   1.08                                                                              1.21                                                                              0.97    1.03 1.26                 0.80                 GLY     2.30   1.42                                                                              0.30                                                                              1.12    0.56 1.78                 0.17                 VK      1.30   1.04                                                                              1.26                                                                              1.12    0.60 1.08                 0.85                 VK-M    0.72   0.89                                                                              9.49                                                                              0.15    0.48 0.64       2    4    3.39                 KA      0.99   1.64                                                                              0.10                                                                              1.16    1.02 1.32                 0.05                 KA-M    1.32   1.01                                                                              1.13                                                                              1.02    0.17 1.58                 1.37                 Z-GP    2.54   1.82                                                                              1.24                                                                              1.44    2.51 1.47                 0.83                 Z-GP-M  2.83   1.94                                                                              1.48                                                                              2.76    2.98 2.68                 0.66                 Z-TP 6.5                                                                              4.21   2.19                                                                              5.70                                                                              2.79    4.73 2.20       5         2.01                 FDA     0.31   1.01                                                                              1.12                                                                              0.92    0.67 0.93                 0.50                 FDA-NAF 0.39   0.72                                                                              0.23                                                                              0.97    0.80 0.96                 0.08                 DCHFMESPMA                                                                            0.05   0.31                                                                              0.38                                                                              0.34    0.14 0.28                 0.13                 GFGA    1.93   0.10                                                                              2.26                                                                              1.85    2.10 2.78                 1.77                 RGES    0.36   1.36                                                                              1.01                                                                              1.62    1.54 1.60                 0.76                 DGLUC   0.22   0.77                                                                              0.98                                                                              1.18    0.13 1.64                 0.38                 DPO4    0.69   0.71                                                                              0.98                                                                              0.88    0.24 1.22                 0.38                 GALAC   2.32   1.01                                                                              3.42                                                                              1.76    1.16 2.35                 1.37                 __________________________________________________________________________

In the above Table the following abbreviations are used:

LEU--(Leu)₂ Rho 110

ALA--(Ala)₂ Rho 110

GLY--(Gly)₂ Rho 110

VK--(Val-Lys)₂ Rho 110

VK-M--(Val-Lys)₂ Rho 110 (modified)

KA--(Lys-Ala)₂ Rho 110

KA-M--(Lys-Ala)₂ Rho 110

Z-GP--(carbobenzyloxycarbonyl-Gly-Pro)₂ Rho 110

Z-GP-M--(carbobenzyloxycarbonyl-Gly-Pro)₂ Rho 110 (modified)

Z-TP6.5--(carbobenzyloxycarbonyl-Thr-Pro)₂ Rho 110 (pH 6.5)

FDA--fluorescein diacetate

FDA-NAF--fluorescein diacetate in a buffer containing sodium fluoride

DCHFMESPMA--dichlorofluorescein in MES buffer plus phorbolmyristateacetate

GFGA--(Gly-Phe-Gly-Ala)₂ Rho 110

RGES--(Arg-Gly-Glu-Ser)₂ Rho 110

DGLUC--(D-glucose)₂ fluorescein

DPO4--(PO₄)₂ fluorescein

GALAC--(D-galactose)₂ fluorescein

The reagents designated as "--M" or "modified" contain cofactors,modulators, inhibitors, etc. as shown in Table 1.

As illustrated from FIG. 16, utilizing only the eigenvector 1 and 2 orthe squared deviation from the means analysis provides the correctdiagnosis, whereas eigenvector 1 alone cannot distinguish from diseases#3 and #4. From Table 5, using three cell types and 18 enzymeconcentrations, it is apparent that only six or eleven values arenecessary to classify the unknown sample. The information mostinformative to the disease diagnosis came from the monocyte cell typeand cathepsin, aminopeptidase and dipeptidyl peptidase enzymes.

EXAMPLE 34 Analysis and Diagnosis of Normal and Diseased States usingArtificial Intelligence

Artificial intelligence was used to analyze data of cellular enzymefunctions, and determine normal and disease states. Peptidases were usedto distinguish leukemia from non-leukemia (output pattern). For inputpatterns lymphocyte and granulocyte cell types were used withaminopeptidases, cathepsins and dipeptidylpeptidase enzyme activities.In this Example, illustrated in Tables 6A-6C, three normals and threeleukemia patients were used as known output patterns for the neuralnetwork to learn (in practice, the larger the learning set the moreaccurate unknown prediction will be). Unknowns were then presented tothe learned algorithm as shown in the test case examples. Clinicaldiagnosis was confirmed by physicians. The trained neural network wasable to correctly classify the leukemia from the non-leukemia.

                  TABLE 6A                                                        ______________________________________                                        THE USE OF PEPTIDASES TO DISTINGUISH LEUKEMIA FROM                            NON-LEUKEMIA                                                                  ______________________________________                                        Aminopeptidases are Pro, Lys, Gly, ala; Cathpsin B and Gln/Ser, Val/Ser       and Leu/gly; Cathepsin C is Thr/.Pro and Dipeptidylpeptidase IV is            Gly/Pro.                                                                      The results of this study shows 100% predictability of leukemia when          tested against normal and various diseases as specified.                      LEARNED CASES:                                                                Substrates: Pro, Lys, Gly, Ala, Gln/Ser, Thr/Pro, Val/Ser, Leu/Gly and        Gly/Pro                                                                       Patients:                                                                              ID #       DIAGNOSIS**                                               ______________________________________                                                 1. N191    Normal                                                             2. N192    Normal                                                             3. N193    Normal                                                             4. J4      Acute Leukemia                                                     5. J5      Acute Myelogenous Leukemia                                         6. J4      Acute Leukemia                                            ______________________________________                                        TEST CASES:                                                                   Patients:                                                                              ID #       DIAGNOSIS**                                               ______________________________________                                                  1. J4     Acute Leukemia                                                      2. J19    Chronic Lymphocytic Leukemia                                        3. J22    Acute Myelogenous Leukemia                                          4. N199*  Normal                                                              5. N200   Normal                                                              6. N201   Normal                                                              7. N206   Normal                                                              8. P68    Abnormal - Tachycardia                                              9. P69    Abnormal - Pancreatic Cancer                                       10. N191   Normal                                                             11. N192   Normal                                                             12. N193   Normal                                                             13. J4     Acute Leukemia                                                     14. J5     Acute Myelogenous Leukemia                                         15. J4     Acute Leukemia                                                     16. P70    Abnormal - Cirrhosis/Hemobilia                                     17. P71    Abnormal - Acute Pyelonephritis                                    18. N194   Normal                                                             19. N195   Normal                                                             20. N197   Normal                                                             21. N202   Normal                                                             22. J22    Acute Myelogenous Leukemia                                ______________________________________                                         *Normal donor later found to have cervical cancer                             **Diagnosis provided by Jackson memorial Hospital; Normal donors were         inhouse employees                                                        

                  TABLE 6B                                                        ______________________________________                                        TEST CASE #4                                                                  ______________________________________                                        Synopsis: Lymphs and Grans were used on Leam and Test cases with the          following results:                                                            SCORE                                                                         I.D. #  Negative Leukemic Diagnosis                                           ______________________________________                                         1  J4               81.4   Acute Leukemia                                     2  J19              100    Chronic Lymphocytic Leukemia                       3  J22              77.9   Acute Myelogenous Leukemia                         4  N199*   63.5            Normal                                             5  N200    100             Normal                                             6  N201    100             Normal                                             7  N206    100             Normal                                             8  P68     100             Tachycardia                                        9  P69     100             Pancreatic Cancer                                 10  N191    100             Normal                                            11  N192    100             Normal                                            12  N193    100             Normal                                            13  J4               100    Acute Leukemia                                    14  J5               99.4   Acute Myelogenous Leukemia                        15  J4               99.3   Acute Leukemia                                    16  P70     100             Cirrhosis/Hemobilia                               17  P71     100             Acute Pyelonephritis                              18  N194    95.6            Normal                                            19  N195    100             Normal                                            20  N197     99             Normal                                            21  N202    98.5            Normal                                            22  J22              72.4   Acute Myelogenous Leukemia                        ______________________________________                                        PREDICTION: 100%                                                                              Non-Leukemic                                                                             Leukemic                                           ______________________________________                                        Dx Non-Leukemic 15/15      0/15                                               Dx Leukemic     0/7        7/7                                                ______________________________________                                         *Normal donor later found to have cervical cancer                        

                                      TABLE 6C                                    __________________________________________________________________________    CLASSIFICATION OF NEW CASES                                                   __________________________________________________________________________    J4 11/21/91                                                                             J5 11/27/91                                                                             P70         P71                                           ACUTE LEUK.                                                                             A M L     CIRHOSIS/HEMOBILIA                                                                        AC. PYELONEPHRITIS                            __________________________________________________________________________    2.72                                                                              GP Lymphs                                                                           3.40                                                                              GP Lymphs                                                                           6.68 GP Lymphs                                                                            3.55                                                                              GP Lymphs                                 0.47                                                                              PRO L 0.89                                                                              PRO L 0.56 PRO L  0.52                                                                              PRO L                                     0.77                                                                              LYS L 5.71                                                                              LYS L 1.02 LYS L  0.91                                                                              LYS L                                     14.02                                                                             GLY L 28.36                                                                             GLY L 54.84                                                                              GLY L  41.93                                                                             GLY L                                     86.00                                                                             ALA L 92.44                                                                             ALA L 141.87                                                                             ALA L  109.26                                                                            ALA L                                     0.05                                                                              QS L  0.07                                                                              QS L  0.04 QS L   0.05                                                                              QS L                                      2.30                                                                              TP L  1.32                                                                              TP L  1.89 TP L   1.86                                                                              TP L                                      0.05                                                                              VS L  0.05                                                                              VS L  0.04 VS L   0.08                                                                              VS L                                      0.16                                                                              LG L  0.23                                                                              LG L  0.79 LG L   0.68                                                                              LG L                                      10.75                                                                             GP Grans                                                                            8.95                                                                              GP Grans                                                                            16.42                                                                              GP Grans                                                                             11.07                                                                             GP Grans                                  5.77                                                                              PRO G 5.92                                                                              PRO G 0.66 PRO G  6.33                                                                              PRO G                                     3.01                                                                              LYS G 14.86                                                                             LYS G 2.33 LYS G  4.17                                                                              LYS G                                     27.79                                                                             GLY G 56.33                                                                             GLY G 88.42                                                                              GLY G  59.35                                                                             GLY G                                     193.43                                                                            ALA G 194.85                                                                            ALA G 236.23                                                                             ALA G  185.37                                                                            ALA G                                     0.22                                                                              QS G  1.24                                                                              QS G  0.19 QS G   0.51                                                                              QS G                                      5.96                                                                              TP G  2.72                                                                              TP G  4.02 TP G   4.37                                                                              TP G                                      0.37                                                                              VS G  1.13                                                                              VS G  0.26 VS G   0.69                                                                              VS G                                      0.85                                                                              LG G  2.51                                                                              LG G  2.20 LG G   4.00                                                                              LG G                                      0.00                                                                              NORMAL                                                                              0.63                                                                              NORMAL                                                                              100.00                                                                             NORMAL 100.00                                                                            NORMAL                                    100.00                                                                            LEUKEMIC                                                                            99.37                                                                             LEUKEMIC                                                                            0.00 LEUKEMIC                                                                             0.00                                                                              LEUKEMIC                                  __________________________________________________________________________    N194      N195      N197        N202                                          NORMAL    NORMAL    NORMAL      NORMAL                                        __________________________________________________________________________    5.22                                                                              GP Lymphs                                                                           5.24                                                                              GP Lymphs                                                                           5.97 GP Lymphs                                                                            6.61                                                                              GP Lymphs                                 0.86                                                                              PRO L 0.88                                                                              PRO L 0.57 PRO L  0.89                                                                              PRO L                                     0.93                                                                              LYS L 1.07                                                                              LYS L 0.70 LYS L  0.71                                                                              LYS L                                     39.76                                                                             GLY L 48.18                                                                             GLY L 42.96                                                                              GLY L  44.55                                                                             GLY L                                     97.02                                                                             ALA L 118.81                                                                            ALA L 96.36                                                                              ALA L  118.37                                                                            ALA L                                     0.02                                                                              QS L  0.04                                                                              QS L  0.06 QS L   0.01                                                                              QS L                                      1.82                                                                              TP L  1.90                                                                              TP L  1.96 TP L   2.49                                                                              TP L                                      0.07                                                                              VS L  0.06                                                                              VS L  0.11 VS L   0.04                                                                              VS L                                      2.06                                                                              LG L  1.93                                                                              LG L  1.79 LG L   1.28                                                                              LG L                                      12.49                                                                             GP Grans                                                                            13.31                                                                             GP Grans                                                                            15.85                                                                              GP Grans                                                                             14.44                                                                             GP Grans                                  3.75                                                                              PRO G 4.70                                                                              PRO G 3.76 PRO G  4.96                                                                              PRO G                                     1.56                                                                              LYS G 2.53                                                                              LYS G 1.59 LYS G  13.22                                                                             LYS G                                     60.30                                                                             GLY G 79.13                                                                             GLY G 76.88                                                                              GLY G  66.22                                                                             GLY G                                     164.00                                                                            ALA G 221.28                                                                            ALA G 188.20                                                                             ALA G  201.17                                                                            ALA G                                     0.07                                                                              QS G  0.21                                                                              QS G  0.24 QS G   0.08                                                                              QS G                                      3.77                                                                              TP G  3.74                                                                              TP G  3.82 TP G   4.44                                                                              TP G                                      0.29                                                                              VS G  0.37                                                                              VS G  0.58 VS G   0.24                                                                              VS G                                      3.52                                                                              LG G  4.11                                                                              LG G  4.41 LG G   2.70                                                                              LG G                                      95.56                                                                             NORMAL                                                                              100.00                                                                            NORMAL                                                                              99.02                                                                              NORMAL 98.62                                                                             NORMAL                                    4.46                                                                              LEUKEMIC                                                                            0.00                                                                              LEUKEMIC                                                                            1.00 LEUKEMIC                                                                             1.32                                                                              LEUKEMIC                                  __________________________________________________________________________    J22 12/9/91                                                                             J4 12/2/91                                                          A M L     A L                                                                 __________________________________________________________________________    3.01                                                                              GP Lymphs                                                                           3.80                                                                              GP Lymphs                                                       0.44                                                                              PRO L 0.29                                                                              PRO L                                                           1.20                                                                              LYS L 1.81                                                                              LYS L                                                           27.14                                                                             GLY L 27.32                                                                             GLY L                                                           83.48                                                                             ALA L 68.10                                                                             ALA L                                                           0.02                                                                              QS L  0.01                                                                              QS L                                                            1.02                                                                              TP L  1.46                                                                              TP L                                                            0.04                                                                              VS L  0.03                                                                              VS L                                                            0.61                                                                              LG L  0.18                                                                              LG L                                                            11.52                                                                             GP Grans                                                                            11.08                                                                             GP Grans                                                        3.53                                                                              PRO G 3.59                                                                              PRO G                                                           1.90                                                                              LYS G 4.09                                                                              LYS G                                                           38.47                                                                             GLY G 47.74                                                                             GLY G                                                           157.48                                                                            ALA G 139.53                                                                            ALA G                                                           0.24                                                                              QS G  0.44                                                                              QS G                                                            2.51                                                                              TP G  2.59                                                                              TP G                                                            0.32                                                                              VS G  0.14                                                                              VS G                                                            1.94                                                                              LG G  0.79                                                                              LG G                                                            27.64                                                                             NORMAL                                                                              0.31                                                                              NORMAL                                                          72.43                                                                             LEUKEMIC                                                                            99.81                                                                             LEUKEMIC                                                        __________________________________________________________________________    J4 12/3/91                                                                              J19 11/27/91                                                                            J22 12/2/91 N99°                                   ACUTE LEUK.                                                                             C L L     A M L       NORMAL                                        __________________________________________________________________________    4.17                                                                              GP Lymphs                                                                           1.73                                                                              GP Lymphs                                                                           1.73 GP Lymphs                                                                            5.55                                                                              GP Lymphs                                 0.24                                                                              PRO L 0.44                                                                              PRO L 0.40 PRO L  1.02                                                                              PRO L                                     1.64                                                                              LYS L 0.98                                                                              LYS L 0.74 LYS L  0.33                                                                              LYS L                                     31.51                                                                             GLY L 11.39                                                                             GLY L 24.50                                                                              GLY L  30.68                                                                             GLY L                                     74.64                                                                             ALA L 47.78                                                                             ALA L 84.42                                                                              ALA L  91.07                                                                             ALA L                                     0.06                                                                              QS L  0.06                                                                              QS L  0.02 QS L   0.02                                                                              QS L                                      1.18                                                                              TP L  0.50                                                                              TP L  0.99 TP L   1.86                                                                              TP L                                      0.04                                                                              VS L  0.01                                                                              VS L  0.01 VS L   0.03                                                                              VS L                                      0.19                                                                              LG L  0.02                                                                              LG L  0.02 LG L   0.85                                                                              LG L                                      1.28                                                                              GP Grans                                                                            1.39                                                                              GP Grans                                                                            13.98                                                                              GP Grans                                                                             13.41                                                                             GP Grans                                  3.68                                                                              PRO G 5.50                                                                              PRO G 4.03 PRO G  4.85                                                                              PRO G                                     3.19                                                                              LYS G 4.41                                                                              LYS G 1.41 LYS G  0.95                                                                              LYS G                                     48.53                                                                             GLY G 48.80                                                                             GLY G 34.02                                                                              GLY G  45.00                                                                             GLY G                                     138.89                                                                            ALA G 143.11                                                                            ALA G 157.12                                                                             ALA G  157.30                                                                            ALA G                                     0.19                                                                              QS G  1.16                                                                              QS G  0.10 QS G   0.05                                                                              QS G                                      1.93                                                                              TP G  2.95                                                                              TP G  2.33 TP G   3.71                                                                              TP G                                      0.23                                                                              VS G  2.83                                                                              VS G  0.22 VS G   0.14                                                                              VS G                                      1.06                                                                              LG G  36.35                                                                             LG G  1.56 LG G   2.022                                                                             LG G                                      18.76                                                                             NORMAL                                                                              0.00                                                                              NORMAL                                                                              22.16                                                                              NORMAL 63.52                                                                             NORMAL                                    81.42                                                                             LEUKEMIC                                                                            100.00                                                                            LEUKEMIC                                                                            77.90                                                                              LEUKEMIC                                                                             36.50                                                                             LEUKEMIC                                  __________________________________________________________________________    N200      N201      N206        P68                                           NORMAL    NORMAL    NORMAL      TACHYCARDIA                                   __________________________________________________________________________    5.38                                                                              GP Lymphs                                                                           6.53                                                                              GP Lymphs                                                                           7.09 GP Lymphs                                                                            5.92                                                                              GP Lymphs                                 0.74                                                                              PRO L 1.10                                                                              PRO L 1.28 PRO L  0.70                                                                              PRO L                                     0.90                                                                              LYS L 1.05                                                                              LYS L 1.65 LYS L  1.51                                                                              LYS L                                     42.28                                                                             GLY L 45.32                                                                             GLY L 55.56                                                                              GLY L  59.29                                                                             GLY L                                     101.38                                                                            ALA L 106.72                                                                            ALA L 122.71                                                                             ALA L  1471.10                                                                           ALA L                                     0.01                                                                              QS L  0.02                                                                              QS L  0.04 QS L   0.02                                                                              QS L                                      2.39                                                                              TP L  1.88                                                                              TP L  2.89 TP L   1.90                                                                              TP L                                      0.06                                                                              VS L  0.07                                                                              VS L  0.28 VS L   0.05                                                                              VS L                                      1.55                                                                              LG L  1.57                                                                              LG L  3.12 LG L   0.78                                                                              LG L                                      12.74                                                                             GP Grans                                                                            15.92                                                                             GP Grans                                                                            17.96                                                                              GP Grans                                                                             16.08                                                                             GP Grans                                  4.04                                                                              PRO G 6.42                                                                              PRO G 6.13 PRO G  7.19                                                                              PRO G                                     1.46                                                                              LYS G 2.06                                                                              LYS G 3.11 LYS G  3.09                                                                              LYS G                                     59.39                                                                             GLY G 75.48                                                                             GLY G 91.93                                                                              GLY G  100.00                                                                            GLY G                                     171.06                                                                            ALA G 206.06                                                                            ALA G 232.57                                                                             ALA G  276.07                                                                            ALA G                                     0.06                                                                              QS G  0.09                                                                              QS G  0.27 QS G   0.33                                                                              QS G                                      4.69                                                                              TP G  4.23                                                                              TP G  5.98 TP G   4.63                                                                              TP G                                      0.24                                                                              VS G  0.32                                                                              VS G  1.03 VS G   0.33                                                                              VS G                                      3.40                                                                              LG G  3.14                                                                              LG G  6.01 LG G   2.52                                                                              LG G                                      100.00                                                                            NORMAL                                                                              100.00                                                                            NORMAL                                                                              100.00                                                                             NORMAL 100.00                                                                            NORMAL                                    0.00                                                                              LEUKEMIC                                                                            0.00                                                                              LEUKEMIC                                                                            0.00 LEUKEMIC                                                                             0.00                                                                              LEUKEMIC                                  __________________________________________________________________________    P69       N191      N192        N193                                          PANCREATIC CA                                                                           NORMAL    NORMAL      NORMAL                                        __________________________________________________________________________    6.80                                                                              GP Lymphs                                                                           4.14                                                                              GP Lymphs                                                                           3.00 GP Lymphs                                                                            3.00                                                                              GP Lymphs                                 0.66                                                                              PRO L 0.69                                                                              PRO L 1.14 PRO L  0.95                                                                              PRO L                                     1.22                                                                              LYS L 1.18                                                                              LYS L 0.56 LYS L  0.52                                                                              LYS L                                     67.38                                                                             GLY L 43.57                                                                             GLY L 37.47                                                                              GLY L  39.30                                                                             GLY L                                     130.34                                                                            ALA L 95.72                                                                             ALA L 103.31                                                                             ALA L  100.50                                                                            ALA L                                     0.31                                                                              QS L  0.02                                                                              QS L  0.03 QS L   0.03                                                                              QS L                                      2.26                                                                              TP L  2.14                                                                              TP L  2.12 TP L   2.33                                                                              VS L                                      0.18                                                                              VS L  0.14                                                                              VS L  0.08 VS L   0.09                                                                              VS L                                      2.00                                                                              LG L  2.42                                                                              LG L  3.45 LG L   1.56                                                                              LG L                                      16.21                                                                             GP Grans                                                                            11.20                                                                             GP Grans                                                                            11.22                                                                              GP Grans                                                                             8.87                                                                              GP Grans                                  5.70                                                                              PRO G 4.34                                                                              PRO G 4.34 PRO G  4.75                                                                              PRO G                                     2.14                                                                              LYS G 1.20                                                                              LYS G 1.20 LYS G  1.43                                                                              LYS G                                     88.04                                                                             GLY G 73.20                                                                             GLY G 43.67                                                                              GLY G  63.33                                                                             GLY G                                     204.02                                                                            ALA G 177.32                                                                            ALA G 154.84                                                                             ALA G  178.64                                                                            ALA G                                     0.37                                                                              QS G  0.19                                                                              QS G  0.15 QS G   0.14                                                                              QS G                                      4.21                                                                              TP G  4.73                                                                              TP G  4.62 TP G   4.96                                                                              TP G                                      0.57                                                                              VS G  0.64                                                                              VS G  0.40 VS G   0.44                                                                              VS G                                      3.53                                                                              LG G  5.37                                                                              LG G  6.78 LG G   3.65                                                                              LG G                                      100.00                                                                            NORMAL                                                                              100.00                                                                            NORMAL                                                                              100.00                                                                             NORMAL 98.77                                                                             NORMAL                                    0.00                                                                              LEUKEMIC                                                                            0.00                                                                              LEUKEMIC                                                                            0.00 LEUKEMIC                                                                             1.23                                                                              LEUKEMIC                                  __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    ABERRANT ENZYME ACTIVITIES IN 10 FEBRILE CHILDREN                             __________________________________________________________________________            LYMPHS                                                                PATIENTS                                                                              P88 P89  P92 P95  P96  P97    P99   P101    P102                                                                             P103                   __________________________________________________________________________     1 Leu  +   +    +   +    +    o      o     +       o  +                       2 Ala  o   o    o             o                    o  o                       3 Pro           o             o                       o                       4 Lys           o             o                       o                       5 Gly  o   -    o             o                    o  o                       6 Ser           o             o                       o                       7 Arg           o   o    -    +      o             o  o                       8 Arg-TFA       o             o                       o                       9 Asp           o             o                       o                      10 Val/Ser       -   o    o    o      +     -       -  o                      11 Val/Ser-M     o             o                       o                      12 Val/Lys                                                                            o   o    o   -    o    o      +     o       o  o                      13 Val/Lys-M                                                                          o   o    o   +    +    +            o       o  o                      14 Gln/Ser       o             o                       o                      16 Gln/Ser-M     o             o                       o                      16 Leu/Gly       o   -    -    o      +     -       o  o                      17 Leu/Gly-M     o             o                       o                      18 Lys/Ala                                                                            o   o    o   o    o    o      o             o  o                      19 Lys/Ala-M                                                                          o   o    o   o    o    o      +             o  o                      20 Z-Ala/Ala     -   -    -    -      o     -       o  -                      21 Z-Ala/Ala-M   -             -                       -                      22 Z-Gly/Pro                                                                          o   -    -   -    -    o            -          -                      23 Z-Gly/Pro-M                                                                        -   -    -             -                       -                      24 Gly/Leu       o             o                       o                      25 Gly/Leu-M     o             o                       o                      26 Ala/Gly                                                                            -   -    -   -    -    -      o             o  o                      27 Ala/Gly-M     -             -                       -                      28 Ala/Ala-TFA   o             o                       o                      29 Ala/Ala-M     o             o                       o                      30 TP 6.5                                                                             -   -    -             o                       o                      31 TP 6.5-M      -             o                       o                      32 LLR           o             o                       o                      33 LLR-M                                                                      34 LGLG          o             o                       o                      35 LGLG-M                                                                     36 FDA  o   +    +   +    +    o      o     +       o  +                      37 FDA-NaF                                                                            o   +    +   +    +    o      o     +       o  +                      38 DCFH o   +    +   +    +    o      o     +       o  +                      40 DCFH.sub.-- PMA                                                                    o   +    +   +    +    +      o     +       o  +                      42 GPLGP                                                                              o   o    -             -                    o  -                      43 GPLGP-M       -             o                       o                      44 GFGA o   o    o             o                       o                      45 RGES o   o    o   o    +    o      o     o       o  o                      46 DGLUC                                                                              o   o    o                                     -                      47 DPO4 o   o    o   o    -    o      o     o       o  o                      45 GALAC                                                                              o   o    o             o                       o                      49 TP 8.7-M      -        o                         o                         50 TP 8.7        -             o                       o                      51 DIGLUC        o             o                       o                              Enteritis                                                                         Viral                                                                              Adenitis                                                                          Viral                                                                              Viral                                                                              Pyelonephritis                                                                       Viral Fever-Unknown                                                                         UTI                                                                              Viral Syndrome                     Stomatitis                                                                             Syndrome                                                                           Syndrome    Syndrome                                                                            Origin                            __________________________________________________________________________            MONOS                                                                 PATIENTS                                                                              P88 P89  P92 P95  P96  P97    P99   P101    P102                                                                             P103                   __________________________________________________________________________     1 Leu  +   +    +   +    +    +      o     +       o  +                       2 Ala  o   o    +             +                    +  o                       3 Pro           o             o                       o                       4 Lys           +             o                       +                       5 Gly  o   o    o             +                       o                       6 Ser           o             o                       +                       7 Arg           +   o    o    o      o             o  +                       8 Arg-TFA       +             o                       +                       9 Asp           o             o                       o                      10 Val/Ser       -   -    o    -      +     -       -  o                      11 Val/Ser-M     -             -                       o                      12 Val/Lys                                                                            -   -    o   o    o    o      +     o       -  +                      13 Val/Lys-M                                                                          -   o    -   o    o    o            -       -  o                      14 Gln/Ser       o             o                       +                      16 Gln/Ser-M     -             -                       o                      16 Leu/Gly       o   -    o    o      +     o          o                      17 Leu/Gly-M     -             o                       o                      18 Lys/Ala                                                                            o   o    o   o    o    +      o     +       o  o                      19 Lys/Ala-M                                                                          -   -    -   -    -    -      o             o  -                      20 Z-Ala/Ala     -   -    o    o      o     -       o  o                      21 Z-Ala/Ala-M   o             o                       o                      22 Z-Gly/Pro                                                                          o   o    -   o    o    o      o     -       o  o                      23 Z-Gly/Pro-M                                                                        -   o    -             o                       o                      24 Gly/Leu       o             o                       o                      25 Gly/Leu-M     -             o                       o                      26 Ala/Gly                                                                            -   -    o   -    o    o      o             o  o                      27 Ala/Gly-M     -             o                       o                      28 Ala/Ala-TFA   +             +                       +                      29 Ala/Ala-M     -             o                       o                      30 TP 6.5                                                                             -   -    -        o                         o                         31 TP 6.5-M      -             o                       o                      32 LLR           o             o                       +                      33 LLR-M                                                                      34 LGLG          o             o                       o                      35 LGLG-M        -             -                       +                      36 FDA  o   +    +   +    +    +      o     +       o  +                      37 FDA-NaF                                                                            o   +    +   +    +    o      +     +          +                      38 DCFH o   +    +   +    +    +      o     +       o  +                      40 DCFH.sub.-- PMA                                                                    o   +    +   +    +    +      o     +       o  +                      42 GPLGP                                                                              -   o    -             -                       -                      43 GPLGP-M       o             +                       +                      44 GFGA o   o    o             o                       o                      45 RGES -   o    +   o    o    o      o     -       o  o                      46 DGLUC                                                                              -   o    -                                     +                      47 DPO4 o   +    +   +         o      o     +       o  +                      45 GALAC                                                                              o   o    o             o                       o                      49 TP 8.7-M      -        o                         o                         50 TP 8.7        -             o                       o                      51 DIGLUC        +             o                       +                              Enteritis                                                                         Viral                                                                              Adenitis                                                                          Viral                                                                              Viral                                                                              Pyelonephritis                                                                       Viral Fever-Unknown                                                                         UTI                                                                              Viral Syndrome                     Stomatitis                                                                             Syndrome                                                                           Syndrome    Syndrome                                                                            Origin                            __________________________________________________________________________            GRANS                                                                 PATIENTS                                                                              P88 P89  P92 P95  P96  P97    P99   P101    P102                                                                             P103                   __________________________________________________________________________     1 Leu  +   +    +   +    +    o      o     +       o  +                       2 Ala  o   o    o             o                    o  o                       3 Pro           o             o                       o                       4 Lys           +             +                       o                       5 Gly  o   o    o             o                    o  o                       6 Ser           +             o                       o                       7 Arg           +   o    o    +      +             o  +                       8 Arg-TFA       +             +                       +                       9 Asp           o             o                       o                      10 Val/Ser       -   -    o    o      +             -  o                      11 Val/Ser-M     o             o                       o                      12 Val/Lys                                                                            -   -    o   -    o    +      +     o       o  o                      13 Val/Lys-M                                                                          -   o    o   +    +    o            o       -  o                      14 Gln/Ser       +             o                       +                      16 Gln/Ser-M     +             o                       o                      16 Leu/Gly       o   -    o    o      +     o       o  -                      17 Leu/Gly-M     -             -                       -                      18 Lys/Ala                                                                            o   o    o   o    o    o      o     +       o  o                      19 Lys/Ala-M                                                                          o   o    o   o    o    o      o             o  o                      20 Z-Ala/Ala     -   -    o    o      o             o  o                      21 Z-Ala/Ala-M   o             o                       o                      22 Z-Gly/Pro                                                                          o        -   -    o    o      o                o                      23 Z-Gly/Pro-M                                                                        -        -             o                       o                      24 Gly/Leu       o             +                       o                      25 Gly/Leu-M     o             o                       o                      26 Ala/Gly                                                                            +   o    o   o    o    o      o             o  o                      27 Ala/Gly-M     -             o                       o                      28 Ala/Ala-TFA   +             +                       +                      29 Ala/Ala-M     o             o                       o                      30 TP 6.5                                                                             -   -    -                                  o                         31 TP 6.5-M      -             o                       o                      32 LLR           o             o                       o                      33 LLR-M                                                                      34 LGLG          o             o                       o                      35 LGLG-M        o             o                       o                      36 FDA  o   +    +   +    +    +      o     +       o  +                      37 FDA-NaF                                                                            o   +    +   +    +    o      +     +       o  +                      38 DCFH o   +    +   +    +    +      o     +       o  +                      40 DCFH.sub.-- PMA                                                                    o   +    +   +    +    +      o     +       o  +                      42 GPLGP                                                                              o   o    -             -                    o  -                      43 GPLGP-M       -             o                       o                      44 GFGA o   o    o             o                       o                      45 RGES o   +    +   o    o    o      o     o       o  o                      46 DGLUC                                                                              o   +    +                                     +                      47 DPO4 o   +    +   o    o    o      o     +       o  +                      45 GALAC                                                                              o   o    o             o                       o                      49 TP 8.7-M      -        o                         o                         50 TP 8.7        o             o                       o                      51 DIGLUC        o             o                       o                              Enteritis                                                                         Viral                                                                              Adenitis                                                                          Viral                                                                              Viral                                                                              Pyelonephritis                                                                       Viral Fever-Unknown                                                                         UTI                                                                              Viral Syndrome                     Stomatitis                                                                             Syndrome                                                                           Syndrome    Syndrome                                                                            Origin                            __________________________________________________________________________     + >0.25 Increase over normal range                                            - >0.25 Decrease over normal range                                            o No change                                                              

The use of neural networks has also been used to reduce a large data set(3 cell types x 45 enzyme assays x 5 or more diseases) to only theimportant cell types and enzyme assays for classification of a disease.This can be visualized by graphing the ratio of the diseases to the meanof the normal of all patients with the disease, as illustrated in FIGS.17A, 17B and 17C to reduce the data set, as illustrated in Table 5, andproviding an HLA score sheet, illustrated in Table 7, showing greaterthan ±25% of the mean normal enzyme activity for that cell type.

EXAMPLE 35 Determination of Disease Progression Using ArtificialIntelligence Based Analysis of Cellular Enzyme Function

Progression of disease during treatment and monitoring a return tonormalcy during treatment are shown in FIGS. 18A-18F and the raw data issummarized in FIG. 18G. The time measurements were monitored bythree-dimensional plotting of cell-type enzyme activity patterns of thesample values and normal values. FIG. 18A illustrates raw data forlymphocyte cells of a particular patient as compared to the normal data.The raw data in FIG. 18A was taken on August 25. FIG. 18B illustratesthe raw data of the diseased patient as compared with normal data onSeptember 6. Similarly, FIGS. 18C-18F illustrate raw data for thediseased patient as compared to normal data on September 21, September26, October 12, and October 13, respectively. The time progressionillustrated in FIGS. 18A-18F clearly indicates by October 13, the rawdata of the "diseased patient" is now virtually identical to the normaldata. The increase in stage or complication with additional diseasestates may also be performed in a manner similar to that illlustrated inFIGS. 18A-18F. All patents and publications referred to in thisapplication are hereby incorporated by reference in their entirety.

The invention has been described with reference to the preferredembodiments. It should be understood, however, that the invention is notso limited, and the scope of the invention should be determined withreference to the following claims, rather than to the foregoingspecification.

We claim:
 1. A method to make an assay compound for assaying theactivity of an enzyme inside a metabolically active whole cell, saidassay compound comprising an indicator group and a leaving group, saidleaving group being selected for cleavage by said enzyme, comprising:a.reacting a compound containing a leaving group selected from the groupconsisting of amino acids, peptides, phosphate esters, saccharides,esters, nucleotides, polynucleotides, nucleic acids, pyrimidines,purines, nucleosides, lipids and mixtures thereof and a blocking group,with an agent to form an intermediate complex containing a leaving groupand a blocking group; b. reacting the intermediate complex with acompound containing an indicator group to form a reaction product,wherein said indicator group is at least one selected from the groupconsisting of rhodamine 110, rhodol, fluorescein and derivativesthereof; c. separating the reaction product from side reaction products,by-products and starting materials; d. removing the blocking group fromthe reaction product to obtain an assay compound having an indicatorgroup and leaving group; and e. purifying the assay compound so thatfluorescence of impurities in such assay compound is less thanautofluorescence of the metabolically active cell.
 2. The method ofclaim 1, wherein the blocking group is at least one selected from thegroup consisting of formyl, acetyl, triftuoroacetyl, benzyloxycarbonyl,phthaloyl, benzoyl, acetoacetyl, chloroacetyl, phenoxycarbonyl,carbobenzoxy, substituted benzyloxycarbonyl, tertbutyloxycarbonyl,isopropyloxycarbonyl, allyloxycarbonyl, methoxysuccinyl, succinyl,2,4-dinitrophenyl, dansyl, p-methoxybenzenesulfonyl, and phenylthio. 3.The method of claim 1 wherein the blocking group is at least oneselected from the group consisting of acetyl, benzyloxycarbonyl,tertbutoxycarbonyl(t-BOC), and 9-fluorenylmethyloxycarbonyl(FMOC). 4.The method of claim 1, wherein said derivatives comprise4'(5')aminorhodamine 110, 4'(5')carboxyrhodamine 110,4'(5')chlororhodamine 110, 4'(5')methylrhodamine 110,4'(5')sulforhodamine 110, rhodol, 4'(5')aminorhodol,4'(5')carboxyrhodol, 4'(5')chlororhodol, 4'(5')methylrhodol,4'(5')sulforhodol, fluorescein, 4'(5')aminofluorescein,4'(5')carboxyfluorescein, 4'(5')chlorofluorescein,4'(5')methylfluorescein, and 4'(5')sulfofluorescein.
 5. The method ofclaim 1, wherein said agent utilized to form an intermediate complex isselected from the group consisting of a substituted carbodiimide,benzotriazolyl-N-oxy-tris(dimethylamino) phosphonium hexafluorophosphateand 1-hydroxybenzo-triazole.
 6. A method to make an assay compound forassaying the activity of an enzyme inside a metabolically active wholecell, said assay compound comprising an indicator group and a leavinggroup, said leaving group being selected for cleavage by said enzyme,comprising:a. reacting a compound containing a leaving group selectedfrom the group consisting of amino acids, peptides, sulfates,phosphates, esters, phosphate esters, nucleotides, polynudeotides,nucleic acids, pyrimidines, purines, nucleosides, lipids and mixturesthereof and a blocking group, with an agent to form an intermediatecomplex containing a leaving group and a blocking group; b. reacting theintermediate complex with a compound containing an indicator group toform a reaction product, wherein said indicator group is at least oneselected from the group consisting of rhodamine 110, rhodol, fluoresceinand derivatives thereof; c. separating the reaction product from sidereaction products, by-products and starting materials; d. removing theblocking group from the reaction product to obtain an intermediatecompound having an indicator group and leaving group; e. reacting theintermediate compound having an indicator group and leaving group withan acid or a base to form a physiologically acceptable salt of saidassay compound for assaying the activity of an enzyme inside ametabolically active which cell; and f. purifying the physiologicallyacceptable salt of said assay compound so that fluorescence ofimpurities in such assay compound is less than autofluorescence of themetabolically active cell.
 7. The method of claim 6, wherein theblocking group is at least one selected from the group consisting offormyl, acetyl, trifluoroacetyl, benzyloxycarbonyl, phthaloyl, benzoyl,acetoacetyl, chloroacetyl, phenoxycarbonyl, carbobenzoxy, substitutedbenzyloxycarbonyl, tertbutyloxycarbonyl, isopropyloxycarbonyl,allyloxycarbonyl, methoxysuccinyl, succinyl, 2,4-dinitrophenyl, dansyl,p-methoxybenzenesulfonyl, and phenylthio.
 8. The method of claim 7,wherein the blocking group is at least one selected from the groupconsisting of tertbutyloxy-carbonyl, 9-fluorenylmethyloxycarbonyl, andacetyl.
 9. The method of claim 7, wherein said derivative comprises4'(5') aminorhodamine 110, 4'(5')carboxyrhodamine 110,4'(5')chlororhodamine 110, 4'(5')methylrhodamine 110,4'(5')sulforhodamine 110, rhodol, 4'(5')aminorhodol,4'(5')carboxyrhodol, 4'(5')chlororhodol, 4'(5')methylrhodol,4'(5')sulforhodol, fluorescein, 4'(5')aminofluorescein,4'(5')carboxyfluorescein, 4'(5')chlorofluorescein,4'(5')methyl-fluorescein, and 4'(5')sulfofluorescein.
 10. The method ofclaim 6, wherein said salt is an acid salt selected from the groupconsisting of hydrochloric, maleic, acetic, trifluoroacetic, tartaricacid, citric, succinic, and p-toluenesulfonic acid or a base saltselected from the group consisting of ammonia and organic bases.